Human Molecular Genetics, 2014, Vol. 23, No.

17 4581–4596
doi:10.1093/hmg/ddu171
Advance Access published on April 9, 2014

Prion protein facilitates synaptic vesicle release
by enhancing release probability
Susan W. Robinson1, Marie L. Nugent1,2, David Dinsdale1 and Joern R. Steinert1,∗
1
MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK and 2Department of Genetics,
University of Leicester, University Road, Leicester LE1 7RH, UK

Downloaded from http://hmg.oxfordjournals.org/ at University of South Dakota and School of Medicine on April 12, 2015
Received March 10, 2014; Revised April 4, 2014; Accepted April 8, 2014

The cellular prion protein (PrPC) has been implicated in several neurodegenerative diseases as a result of protein
misfolding. In humans, prion disease occurs typically with a sporadic origin where uncharacterized mechan-
isms induce spontaneous PrPC misfolding leading to neurotoxic PrP-scrapie formation (PrPSC). The conse-
quences of misfolded PrPC signalling are well characterized but little is known about the physiological roles
of PrPC and its involvement in disease. Here we investigated wild-type PrPC signalling in synaptic function as
well as the effects of a disease-relevant mutation within PrPC (proline-to-leucine mutation at codon 101).
Expression of wild-type PrPC at the Drosophila neuromuscular junction leads to enhanced synaptic responses
as detected in larger miniature synaptic currents which are caused by enlarged presynaptic vesicles. The ex-
pression of the mutated PrPC leads to reduction of both parameters compared with wild-type PrPC. Wild-type
PrPC enhances synaptic release probability and quantal content but reduces the size of the ready-releasable ves-
icle pool. Partially, these changes are not detectable following expression of the mutant PrPC. A behavioural test
revealed that expression of either protein caused an increase in locomotor activities consistent with enhanced
synaptic release and stronger muscle contractions. Both proteins were sensitive to proteinase digestion. These
data uncover new functions of wild-type PrPC at the synapse with a disease-relevant mutation in PrPC leading to
diminished functional phenotypes. Thus, our data present essential new information possibly related to prion
pathogenesis in which a functional synaptic role of PrPC is compromised due to its advanced conversion into
PrPSC thereby creating a lack-of-function scenario.

INTRODUCTION disease may now be caused by either loss-of-function of PrPC
or gain-of-function of cytotoxic PrPSC, or both.
The cellular prion protein (PrPC) is a cell membrane-anchored PrPC is present in all mammalian cortico-cerebellar, deep
glycoprotein which plays an important role in a variety of nuclei neurons and neuromuscular junctions (NMJs) (5). Mor-
neuronal processes including circadian rhythm, neuroprotection phological studies suggest that PrPC is preferentially located
and neuroplasticity (1,2). Although the physiological role of along axons and in presynaptic terminals (6) but postsynaptic lo-
PrPC remains elusive, the conversion of PrPC into the neurotoxic calization and signalling has also been reported (7,8). Evidence
PrPSC during prion disease and its signalling are well documen- accumulates that neuroprotective roles of PrPC are essential
ted (2 – 4). As a consequence of protein misfolding, several mam- (9,10) as loss-of-function in PrPC knock-out (KO)/mutant
malian species develop neurodegenerative conditions best models leads to neuronal dysfunction (11– 13). Interestingly,
known as scrapie in sheep, bovine spongiform encephalopathy KO animals for the gene encoding PrPC exhibit phenotypes
in cattle or Creutzfeldt – Jacob disease (CJD) and Gerstmann – with impaired long-term potentiation (14 –16), abnormal circa-
Stra¨ussler – Scheinker Syndrome (GSS) in human. The unique dian rhythm (17) or effects on glutamatergic transmission
feature of these conditions is that, in addition to sporadic and (18,19) but also more severe characteristics such as Purkinje
inherited forms, it can be transmitted by infectious agents cell degeneration and demyelination of peripheral nerves
according to the ‘protein only’ hypothesis. The early onset of leading to ataxia (11,20). As the exact cellular functions of


To whom correspondence should be addressed. Tel: +44 1162525216; Fax: +44 1162525616; Email: js333@le.ac.uk

# The Author 2014. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4 .0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

have been employed (21– 23) and in particular.oxfordjournals. 1A. ANOVA). 17 PrPC remain unknown. P . Using the more sensitive Kolmogorov – developmentally younger larval stages. it is essential to characterize the physio. prion protein induced larger mEJCs relative to their respective sion of a mutated PrPC (P101L) [PrPP101L].4582 Human Molecular Genetics. 2015 ation of acetylcholine release at the mouse NMJ (32). 23. Each UAS control is linked to the human prion disease GSS. 1C. PrPSC in adult Drosophila causing neurodegeneration and ex. studies have yet to define the exact physiological mechanisms of PrPC in order to explain the underpinning synaptic loss and/ or dysfunction before disease onset.30). In initial experiments for PrP3F4 or PrPP101L did not differ from WT (w1118) controls we aimed to validate expression of either PrP3F4 or the mutated (UAS-PrP3F4: 0.07 nA (n ¼ 8) versus female: 0.82 + boutons of the NMJ and lack of expression in UAS controls 0.80 + 0. ANOVA). an indicator of PrPSC misfolding. The data show (n ¼ 48).05 each.22. Murine prion protein enlarges synaptic vesicles In the current study.84 + 0. P . Student’s t-test) consistent with no [Fig. co-stained for vesicular glutamate transporter sex-dependence. Any digestion occurring below 5–7 mg/ml logical and neuroprotective roles of PrPC in order to better under. tion of these proteins may not be affected (12).74 + 0. .36) but it is unknown whether this resistance functional properties between both prion proteins within the to PK digestion. although to a lesser degree RESULTS then caused by the PrP3F4 expression (0.001 versus WT controls and P . Student’s t-test). P ¼ 0.05. analysis of variance that endogenous PrP3F4 facilitates synaptic release and this func. Thus. normal- lines (prion protein/a-tubulin ratio: PrP3F4: 1.04 nA (n ¼ 36). 2014. pared with wild-type w1118 (WT) controls [WT: 0. whereas mechanisms for this dysfunction are and whether pre.71 + prion protein (PrPP101L) in transgenic Drosophila larvae by 0.07 nA.00001. PrPC can convert into being digested by this concentration of PK. which induces a GSS-like disease in mice and is Voltage Clamp (TEVC) experiments were conducted to record related to a human GSS-associated mutation (P102L) (35)] miniature excitatory junctional currents (mEJCs). 0. P ¼ 0. Vol. No.001.03 nA tributing to our understanding of PrPC function. P ¼ 0. 0. PrP3F4 ex- was investigated to elucidate potential effects on synaptic pression induced a 38% increase in mean mEJC amplitudes com- release before manifestation of neurodegeneration thereby con. protein misfolding and aggregation is unlikely to occur at this de- pression of a mutated PrPC (PrPP101L) is sufficient to mimic velopmental stage (12). n-number of NMJs indicated in bars).024. Comparison of mEJC amplitudes performing immunohistochemistry (IHC) which confirmed in WT controls of male and female larvae did not reveal any dif- strong and specific expression of either protein within all ference (male: 0. P ¼ 0. The use of w1118 as con- tion is partially compromised following expression of PrPP101L trols in addition to respective UAS controls was justified as all indicating a pivotal role of PrPC (PrP3F4) signalling. K – S test) suggesting that 5 –7 mg/ml (30. PrP3F4: 1. 0. 2A and B]. PrP3F4 versus flies (30 days) expressing PrP3F4 where digestion occurs above PrPP101L: D ¼ 0. The current study suggests that synaptic dysfunction precedes the cell death that aims to investigate prion protein-mediated effects on presynap- occurs at later stages during prion pathogenesis (33. In order to test this we Smirnov test (K– S test) a difference between the distributions employed the Proteinase K (PK)-digestion protocol. Assessing expression levels normalized histograms and cumulative histograms for the of both prion proteins revealed no differences between both three different genotypes. Incubation of Recently. As shown in Figure 2C and D. presynaptic expression in Drosophila of To test the effects of PrP3F4 (pan-neuronal expression) on the mouse wild-type PrPC (PrP3F4) and a mutated form of PrPC synaptic physiology at Drosophila NMJs. Western blot analysis further confirmed expression shift in the distribution of single quantal size as depicted in the of either prion protein (Fig. increased resistance to PK digestion has been shown in older PrPP101L versus Ctrl: D ¼ 0. UAS-PrPP101L: 0.30) we next tested whether the expressed tude distributions in PrP3F4 but to a lesser degree in PrPP101L prion proteins were sensitive to proteinase digestion at these expressing larvae. The increase in mEJC amplitudes is due to a (vGlut)]. P .30. 1B). This augmentation in mean mEJC amplitudes was also observed at NMJs expressing PrPP101L (23%). male larvae containing one copy of either Expression of wild-type murine PrPC (PrP3F4) in Drosophila UAS-PrP3F4 or UAS-P101LD PrPC [PrPP101L] were used (from causes spongiform degeneration in adult fly brains (26) and now on referred to as UAS controls). soma (34) but it remains to be investigated what the underlying Downloaded from http://hmg.36.0 + 0. Fig.05 versus PrP3F4. murine prion disease models precedes degeneration of the cell PrPC can modulate synaptic transmission (31) including potenti.or postsy- PrPC-KO mice exhibit reduced inhibitory release (14). 0. a mutation which UAS controls (P . As frequency histograms demonstrated an increased probability of PrPC expression induces a neurodegenerative phenotype in larger amplitude events and hence a right shift in mEJC ampli- older adult flies (26. expression of 0– 1 mg/ml) did not indicate any formation of PK-resistant PrPC and PrPSC in Drosophila or Caenorhabditis elegans allows prion in either PrP3F4 or PrPP101L transgenic lines as PrP3F4 investigations of prion function in host organisms that do not and PrPP101L digestion starts at 1 mg/ml (so is a-tubulin have a direct prion ortholog (24– 29).42.00023.35.2 (n ¼ 7).34) but tic release mechanisms to elucidate its physiological roles. is evident in presynaptic terminal are different.org/ at University of South Dakota and School of Medicine on April 12.02 + 0. 0.91 + 0. 1C). several non-mammalian neurodegeneration models larval preparations with various concentrations of PK (Fig. ized relative histograms for mEJC amplitudes and cumulative PrPP101L: 1. lines were backcrossed to w1118 for at least six generations. An became evident (PrP3F4 versus Ctrl: D ¼ 0.04 nA. could suggest a lack of misfolding and that the putative func- stand the changes which occur during early onset prion disease. Fig. Research naptic prion protein signalling is involved.04 nA (n ¼ 35). It is established that synaptic loss in neurodegenerative phenotypes in adult Drosophila (25. 0. Two-Electrode [PrPP101L. P . Expressed wild-type and mutated murine prion proteins ANOVA.07 nA (n ¼ 5).2 + 0. third instar larvae. Expression of either importantly this degeneration is accelerated following expres. As addi- are sensitive to proteinase digestion tional controls. (ANOVA).4 (n ¼ 10).

Human Molecular Genetics. PK completely digested prion protein (25 kDa bands) at relatively low concentrations (1 mg/ml) and so is a-tubulin being digested. (B) Protein extracts of Tg-PrP lines (elav-Gal4/UAS-MoPrP3F4 and elav-Gal4/UAS-MoPrPP101L) with appropriate controls (UAS-MoPrP3F4/+ and UAS-MoPrPP101L/+) were probed for PrP (Ab: AH6 anti-PrP) and a-tubulin. 2015 Figure 1. n ¼ 5 larval heads per lane. 23.oxfordjournals. n ¼ 3 animals per lane.org/ at University of South Dakota and School of Medicine on April 12.98) which are not detectable in larvae. PrPC can undergo glycosylation leading to multiple PrP bands (25. (C) Third instar larval extracts from Tg-PrP lines (elav-Gal4/UAS-MoPrP3F4 and elav-Gal4/UAS-MoPrPP101L) treated with a mild PK gradient (0– 1 mg/ml) showed no PK resistance. No. Prion protein expression does not result in PK resistance at the larval NMJ. . (A) IHC staining of third instar NMJs show strong prion protein labelling in elav-Gal4/UAS-MoPrP3F4 and elav-Gal4/UAS-MoPrPP101L larvae but no prion expression in respective UAS control NMJs. Note. 17 4583 Downloaded from http://hmg. 2014. Vol.

42. n ¼ 4– 8 animals for UAS controls]). 2014.1 ms. P . versus DGluRIIB contributions (37. 0. This PrP3F4-induced increase in mEJCs could either result release or basal release probability have not changed in response from larger presynaptic vesicles or altered postsynaptic to prion protein expression.00001). 23.22. P . 17 Downloaded from http://hmg. P .38)] were compared. These data suggest that the responses to a released vesicle.4584 Human Molecular Genetics.2 + 0.2 s21. number of synapses and AZs and size of .001.7 + 0.05. tPrP : 6. (D) Relative cumulative amplitude histograms illustrate the right-shift in amplitudes for PrP3F4 expressing larvae. P ¼ 0. the frequency (f) of mEJCs was not different made as shown before (41). Distributions for UAS controls are not shown for simplicity. P ¼ 0. As the Drosophila NMJ harbours two different kinds of that DGluR composition had not changed (tCtrl: 6.2 s21. f3F4 PrP ¼ representative 1s and 1b boutons with release sites [long 1.4 + 0. 0. indicating sizes.org/ at University of South Dakota and School of Medicine on April 12. kinetics of mEJC decays [indicative for DGluRIIA lar release. There is no evidence for multi-quantal release as distributions do not exhibit multiple peaks. ANOVA with Tukey– Kramer post hoc test. 0.oxfordjournals. Data denote mean + SEM. PrP3F4 versus PrPP101L: D ¼ 0. Bouton types were number of functional synapses responsible for Ca2+-independent identified by size.05. ∗∗∗ P .40) suggesting that the fication images for genotypes indicated. n ¼ 6 –48 NMJs. No.00023. TEVC recordings were performed at the NMJ to investigate the effects of neuronal PrP3F4 or PrPP101L expression. ANOVA) and arrows: active zones (AZs)] and vesicles and higher magni- comparable with published data (39.05. PrPP101L expression induced a left-shift relative to PrP3F4 (K–S test.024. Prion causes enlarged miniature EJCs.1 + 0. a subdivision of EM images into 1s and 1b boutons was Further to this. sizes. 0.30. namely 1s and 1b. ANOVA).5 + 0. ∗ P . The images in Figure 3A illustrate between the three genotypes (fCtrl ¼ 1. PrPP101L versus w1118: D ¼ 0. n ¼ 16 animals for PrP PrP3F4. (B) Mean mEJC amplitudes are increased following PrP3F4 and PrPP101L expression with PrPP101L showing a reduction relative to PrP3F4 (n—number of NMJs is indicated in bars.6 + 0. PrPP101L expression left-shifted amplitudes relative to PrPC but still caused an increase versus control (w1118). Vol. boutons. n ¼ 15 animals for PrPP101L. 0.1 s21. (A) Sample traces showing mEJCs in control (Ctrl [w1118]) and PrP3F4 expressing larvae. (C) Normalized amplitude histograms for mEJCs show a right-shift of amplitudes following PrP3F4 expression. [n ¼ 19 animals for w1118.1 ms. both exhibiting different vesicular t3F4 P101L PrP : 6. Mean To further investigate synaptic vesicles at NMJ synapses we decay tau (t) values did not differ between genotypes nor used electron microscopy (EM) to measure presynaptic vesicle did the t distributions between tested conditions. fP101L PrP ¼ 1. 2015 Figure 2. PrP3F4 versus w1118: D ¼ 0.1 ms. To test whether postsynaptic prion protein-mediated changes are not due to postsynaptic Drosophila glutamate receptor (DGluR) have functionally alterations but most likely due to presynaptic changes in vesicu- changed.

(A) EM images for genotypes indicated. The enveloping sub-synaptic reticulum is more voluminous around type 1b boutons.4 to 42.7 to 54.4 + 0.5 + 0. 2015 Figure 3.5 to 43. active zones and mitochondria compared with 1s.1 + 0.4 nm [1b]∗ .oxfordjournals.0 + 1. Images of synaptic boutons showing synaptic active zones (AZ—arrows with T-bars in left two images) and synaptic vesicles at higher magnification (SV—right three images for genotypes indicated).01. 0. Data denote mean + SEM. (D) Left. 23.6 nm [1b]∗ .8 nm [1s]∗ and 38. ∗ P . The boutons have been identified by the following features: 1b boutons are larger and possess more synapses.7 + 0. PrPP101L: 43. mean vesicle diameters for 1s and 1b boutons from elav-Gal4/UAS-MoPrPP101L and control UAS-MoPrPP101L/+ larvae. (C) Mean diameters of total vesicle counts including both 1s and 1b boutons (with similar percentage of both bouton types) for genotypes indicated showing increases of mean vesicle diameters following PrPP101L and PrP3F4 expression. Right. Note.05. No.7 + 0. 0. ∗∗ P . Prion causes enlarged presynaptic vesicles. PrP3F4 caused a greater increase relative to PrPP101L. (PrP3F4: 46. 2014.org/ at University of South Dakota and School of Medicine on April 12.1 nm [1 s]∗ and 37. mean vesicle diameters for 1s and 1b boutons from elav-Gal4/UAS-MoPrP3F4 and control UAS-MoPrP3F4/+ larvae. . Human Molecular Genetics.7 to 47. n—number of boutons is indicated in bars [n ¼ 3 animals for each genotype]. 17 4585 Downloaded from http://hmg. No difference in the mean number of AZ and T-bars between genotypes was detected. Vol.3 + 0.2 + 0. Student’s t-test). (B) Relative average and cumulative histograms of total vesicle diameter counts including both 1s and 1b boutons (with similar percentage of both bouton types) in elav-Gal4/UAS-MoPrPP101L (left) and elav-Gal4/UAS-MoPrP3F4 (right) larvae with their respective UAS controls.

Together. ∗∗ P . total diameters in PrPP101L (n ¼ 16– 17 boutons) and PrP3F4 To discriminate between these two options we initially ana- larvae (n ¼ 9 – 17 boutons) relative to their UAS controls but lyzed paired pulse ratios (PPR) which critically depend on the also the difference between PrPP101L and PrP3F4 expressing initial release probability (pvr) (43) but also on the degree of larvae (∗ P . 2014. increased in PrP3F4 NMJs (w1118 Ctrl: 119 + 6 (n ¼ 12). more.4586 Human Molecular Genetics. versus PrP3F4: D ¼ 0. 17 the sub-synaptic reticulum (41) (Fig.0001.05. Student’s t-test. Further.05.001. in that the amplitude ratio of two teristics (number of synapses and AZs and size of the closely spaced EJCs (EJC2/EJC1) increases as pvr decreases (45). respectively. 0. 0. 0. In of either prion protein led to an increase in mean vesicle diameter all further experiments we used only w1118 larvae as controls in both types of boutons compared with their UAS controls since responses in UAS-PrP3F4 controls and UAS-PrPP101L con- (Fig. PrP3F4: 162 + 11∗ (n ¼ 18). P . as eEJCs decayed with identical time constants at the beginning Evoked EJC (eEJC) amplitudes at low frequency stimulation and end of the train (data not sown). The overall shift in In agreement with previous data. Figure 3B shows (data not shown) again confirming no alterations in postsynaptic average relative cumulative frequency histograms and histo. Stimulation at 50 Hz for 500 ms gesting a differential effect on synaptic transmission (Fig. PrPP101L boutons shows also significant differences (PrPP101L UAS-PrP3F4: 121 + 5 (n ¼ 10). receptor desensitization was excluded NMJs with presynaptic expression of PrP3F4 or PrPP101L. 3D. Inter. 0. 23. The data also imply As changes in the observed PPR between control and PrP3F4 that the mutated form of prion protein (PrPP101L) exhibits a expressing synapses could be caused by alterations of two differ- diminished and altered function compared with PrP3F4 signal. As these facilitation (44) or on receptor saturation and desensitization values are composed of 1s and 1b type boutons (equal number (45. K– S test. To further investigate these PrP3F4 expression did not allow potentiation within the train. A method to determine pool size was successfully applied at Drosophila NMJs by analyzing cumulative amplitudes within trains of higher frequency stimulation (42. namely: (i) the initial pvr of the synapse but also ling. 0. the difference between vesicle diameters from PrP3F4 and PrPP101L: 140 + 8 (n ¼ 5). the ratio of two eEJCs with inter-spike-intervals (ISI) 1b boutons of 45 and 38 nm.47.0001. 0. We sub. summate linearly in eEJCs and mEJCs and evoked responses ability of larger diameters and hence can explain a right-shift arise from the same pool of vesicles at these low stimulus fre- in mEJC amplitude distributions in PrP3F4 and PrPP101L expres. 0. 0. n ¼ 9 – 17 boutons). quantal content [QC.49).0001). Assuming that single quanta grams for vesicle diameters demonstrating an increased prob. Receptor desensitization was excluded as eEJCs of each bouton type). n ¼ 4 – 10 boutons). P . we decided next to test whether prion decay kinetics were identical between the first and last eEJC protein effects could be observed at both bouton types. whereas PrPP101L PrPP101L again showed a non-significant tendency towards an mutants showed an increase of 30 and 41% in 1s and 1b increased pvr. quencies. boutons. Fig. 0. whereas PrP3F4 expressing larvae showed significant directly or indirectly involved in regulating vesicle size. Fig. aptic function and vesicular release.191. 2).46). ANOVA. P . respectively (41). Figure 3C illustrates the increase in mean per vesicle. these parameters (Fig.5 mM [Ca2+]e. The average data of peak (0. 2015 shown above (Fig. the number of AZs per bouton type did not differ strains were back-crossed for at least six generations to w1118. control larvae showed weak the distribution of synaptic vesicle diameter in 1s and 1b paired-pulse facilitation at 100. Vol. 3A). No. Figure 5A Ca21-dependent evoked release is enhanced by prion shows 50 Hz trains (500 ms) of a control and PrP3F4 expressing protein expression NMJ. between the genotypes (data not shown). 40 and 20 ms ISI (Fig. this data suggest that PrP3F4 has an endogenous (ii) the size of the ready-releasable pool (RRP) (43.2 Hz) were enhanced following PrP3F4 (134 + 5 nA∗∗∗ amplitudes revealed the difference between the genotypes as (n ¼ 19). 4B). 4A revealed a smaller cumulative eEJC amplitude (extrapolation and B). 4C) on the changes in vesicular diameter we calculated the increase suggesting an increase in pvr in PrP3F4 larvae and consistent in volume which increased in PrP3F4 expressing larvae by 60 and with increases in QC following PrP3F4 expression (Fig. ∗ P . sub-synaptic reticulum) and vesicle diameter values for 1s and Here.48). 50% in 1s and 1b boutons.05. Control recordings show a strong initial potentiation fol- The above data suggest a functional role of prion protein in syn.01. P . To check whether the observed differences (110 + 9 nA (n ¼ 7)) compared with w1118 Ctrl (101 + 5 nA are due to changes in pvr and/or size of RRP we estimated (n ¼ 18)) and UAS-PrP3F4 controls (103 + 2 nA (n ¼ 10)) sug. 4C) boutons towards larger values suggests that PrP3F4/PrPP101L is (42). effects on release mechanisms we measured evoked release at Under these conditions. 4B). eEJCs did not differ in their decay kinetics to time point zero) of 162 + 32 nA∗∗∗ (n ¼ 8) in PrPP101L . it is function in vesicle biogenesis and positively controls vesicle important to estimate both of these synaptic parameters. within a 1 s train (data not shown). P . Student’s t-test). The QC was strongly D ¼ 0. size and transmitter release at the Drosophila NMJ. Expression varying from 20 to 200 ms was assessed at 1.oxfordjournals.05. Mean total vesicular The enhanced QC could be attributable to an increase in either sizes provide the best comparison to the mean mEJC amplitudes the number of release-ready vesicles or the release probability Downloaded from http://hmg. receptor composition (37). trols did not differ from w1118 (compare Figs 2 and 4B) and all estingly. lowed by depletion which plateaued after 300 ms. the number of vesicles released sing larvae relative to their respective controls (PrP3F4 versus per action potential (AP)] was estimated as the quotient of UAS Ctrl: D ¼ 0.131.org/ at University of South Dakota and School of Medicine on April 12. ANOVA) but not PrPP101L expression shown in Figure 5B. ent parameters. In contrast. These increases are comparable with data from electrophysiological recordings although one has to consider an overestimation of measured mEJC amplitudes due Prion protein reduces synaptic vesicle pool size to non-detectable smaller mEJCs (42). PrPP101L versus UAS Ctrl: eEJC and mEJC amplitude per NMJ. Based depression at various ISI tested (ANOVA. divided the boutons according to previously published charac. PPR provide a measure of pvr. respectively. P . 5C –F).137.

The comparison between the three genotypes pared with 324 + 45 nA (n ¼ 7) in control larvae (ANOVA.05. 17 4587 Downloaded from http://hmg. Data denote mean + SEM. revealed that pvr is increased following expression of PrP3F4 Fig.02 . n ¼ 4 animals for PrPP101L. PPRPrP : 0.12 (n ¼ 8). we further estimated the initial pvr which from parabolic fits to the variance –mean plots for each cell can be calculated based on the cumulative amplitude analysis (Fig. PrP3F4: 1.07 + 0.02.03. eEJCs were elicited at varying calcium concentration (0.03 . By plotting the mean QC over various [Ca2+]e by dividing EJC0 of the train by the size of the RRP as described (Fig. confirming 0. Fig. Black arrows indicate measurements of eEJC amplitudes. was therefore reduced from control of 382 + 44 (n ¼ 7) to We next decided to apply a different and independent ap- 174 + 30∗∗ (n ¼ 13) in PrP3F4 expressing larvae but not in proach to estimate the synaptic parameters by which prion PrPP101L (198 + 32 (n ¼ 8).91 + 0. example traces for PPR for a Ctrl (w1118) and PrPC NMJ at 20 ms inter-stimulus-intervals (ISI) at 1.14∗∗ (n ¼ 13). PPR3F4 P101L 3F4 P101L PrP : 0. PPRPrP : 0. pvr and q were estimated Using this method. PPRPrP : 0. The but not PrPP101L in agreement with above data (Ctrl: 0.02. analysis could be excluded as it was shown previously that recov.001.03.2 Hz. PPRPrP : 0. n ¼ 7 animals for PrPC]).94 + 0. 0. n ¼ 13 [PrP3F4] NMJs [n ¼ 10 animals for controls.01 + tive amplitude by the mean quantal size of each muscle. The dotted line indicates a PPR ∗ ∗ of 1 (20 ms: PPRctrl: 1. 2015 Figure 4.oxfordjournals. Evoked synaptic release and QC are enhanced by PrP3F4 expression. pvr and quantal size q. summary of mean PPR versus ISI between 20 to 200 ms for three different genotypes. 5D and F. PPRPrP : 0. PrP3F4 versus Ctrl: ∗ P .97 + 0.88 + 0.03. 23. 5C and E) suggesting a strong reduction in pool size. ANOVA). 5E). 0.5 mM [Ca2+]e.5 – ery occurs earliest at times . (B) Summary of mean eEJC amplitudes and QC for different genotypes at 1. protein modulates synaptic release: fluctuation analysis (51– bility that recovery from depression was interfering with this data 53) estimates the quantal parameters: N.91 + 0. (C) Left. Fig. n ¼ 5 animals for PrPP101L.89 + 0.01. Fig.34 + pool size was subsequently calculated by multiplying the cumula.04 + 0. PPRPrP : 0. ANOVA with Tukey–Kramer post hoc test. ∗ P .01. ANOVA with Tukey– Kramer post hoc test. 2014.04. 6A and B) and N. 5F. n ¼ 15 animals for PrP3F4.org/ at University of South Dakota and School of Medicine on April 12.92 + 0. 0. 3 mM. The number of release-ready vesicles was calculated decreased the size of the RRP.05. previously (50). Human Molecular Genetics. No. whereas PrPP101L only induced a by dividing the cumulative amplitude by the quantal size and decrease in the RRP size without affecting pvr. n ¼ 5 –19 NMJs (indicated in bars [n ¼ 10 animals for controls. Vol.98 + 0. The possi. n ¼ 6 animals for UAS-PrP3F4]). 6D). and 133 + 20 nA∗∗∗ (n ¼ 13) in PrP3F4 expressing larvae com. Right. 40 ms: PPRctrl: 1.05 at different ISI.20 s (42). (A) representative eEJC recordings by stimulating the motor nerve in genotypes indi- cated at 1. a strong reduction following PrP3F4 but also PrPP101L expression The above data consistently show that PrP3F4 enhanced pvr and (Fig. ANOVA). n ¼ 7 [PrPP101L]. 200 ms: PPRctrl: 0. ∗∗∗ P .04 (n ¼ 7). PrPP101L: 0. 3F4 ∗ P101L 3F4 P101L 100 ms: PPRctrl: 1. 0.5 mM [Ca2+]e.92 + 0. 0.01 . 6C) it became apparent that PrP3F4 expression led to .74 + 0.5 mM [Ca2+]e.04 + 0. PPRPrP : 0. ANOVA (n ¼ 21 [Ctrl].

whereas PrPP101L (EC50. 0.001). sensitivity to Ca2+ was not altered (54). ANOVA). 0. Hill slope was reduced in PrP3F4 versus PrPP101L and control The fluctuation analysis further revealed.5– 2 mM [Ca2+]e. 2). A lower Hill slope has ∗∗ P . Data denote mean + SEM. 0.3 [PrP3F4] (PrP3F4 versus Ctrl: ∗ P . 0. n ¼ 7.01. ∗ P . Vol.3 [PrPP101L] to 2. 0.56) opening up the possibility for an prion protein expressing and control larvae.01 versus affecting the half-maximal effective Ca2+ concentrations Ctrl and P .4588 Human Molecular Genetics. Data were fitted with a Hill equation been associated with a tightening of the release site/Ca2+ yielding the Hill slope as a measure of Ca2+ co-operativity for channel complex (55.001) and vesicle pool sizes (open bars. PrP3F4 versus PrPP101L (731 + 8 pA. mean QC during the 50 Hz stimulus train in the three genotypes indicated. 0. Line fits to the cumulative eEJCs were back-extrapolated to time zero (indicated in italics.3 + 0. n ¼ 9 animals for PrP3F4]). The release probability was estimated by dividing eEJC0 by the pool size.05.5 mM [Ca2+]e. n ¼ 5 animals for PrPP101L. Interestingly. ANOVA with Tukey– Kramer post hoc test (n ¼ 7 –13 NMJs as indicated in bars [n ¼ 4 animals for controls.05. see Materials and Methods).05.05 versus PrPP101L) expressing larvae compared . in agreement with data NMJs from 3. 23. 0. 17 Downloaded from http://hmg. (C) Cumulative eEJC amplitudes for the three genotypes with linear fits to the last 200 ms for each condition. Fig. (B) Mean eEJC amplitudes from synaptic trains for the three different genotypes. P . the interaction of PrP3F4 with Ca2+ signalling. Number of readily releasable vesicles is reduced following PrP3F4 expression.4 + from mEJC measurements (Fig. P .01) and the release probability for each genotype (open bars. enhanced release at 0.001. ∗∗∗ P . No. (F) Mean number of ready-releasable vesicles (black bars. 0.01) expression and even PrPP101L: ∗∗∗ P . ∗∗ P . mean + SD. n ¼ 5. 0.05. The number of vesicles was estimated by dividing pool size by mean quantal size.oxfordjournals. 2014. Inset. n ¼ 8 – 11 NMJs) indicating that expression only enhanced QC at 1 mM [Ca2+]e (∗ P . 6) without further increased in PrP3F4 (886 + 24 pA.3 + 0. 2015 Figure 5. ∗∗ P .01) are reduced compared with control. 0. (D) Average cumulative QCs with back-extrapolation of linear fits to the last 200 ms yielded estimates for the readily releasable pool (indi- cated in italics) for each genotype. 0.5 [Ctrl] and 4. Pool size was determined by multiplying the cumulative eEJC amplitudes with the mean quantal size for this cell. that q was enlarged following 0. 0. ANOVA.org/ at University of South Dakota and School of Medicine on April 12. ∗∗ P . (E) Mean values for cumulative eEJC amplitudes (black bars. (A) Example traces of 50 Hz trains (500 ms) from a Ctrl (black) and PrP3F4 expressing (red) larva at 1. P . ANOVA. 0.

cumulative postsynaptic agreement with above data (Figs 2–4). n ¼ 4 animals for PrPP101L. both. 0.036).9 + 0.0 + 0. . Fur.05.01. 6E middle) ance with previously reported EM data showing a number of consistent with the enhanced QC and decreased PPR in PrP3F4 synapses onto muscle 6 of 500 (57). The estima- not PrPP101L expressing larvae although once again. On the other hand. PrP3F4 had a stronger phenotype then PrPP101L also in P . these estimations expressing larvae (Figs 4 and 5). data denote mean + SD. n ¼ 8 –11 NMJs [n ¼ 5 animals for control.3 for Ctrl. 17 4589 Downloaded from http://hmg. Human Molecular Genetics. Ca2+ dependency is decreased in PrP3F4 but not PrPP101L: controls (3. ANOVA). 0.06 mM and Hill slope ¼ 3. (B) eEJC amplitudes of a PrP3F4 expressing NMJ elicited at 0. Therefore. 4 and 6) were conducted.4).5. with controls (347 + 27 pA. data denote mean + SEM). Prion expression induced changes in synaptic release parameters. n ¼ 5 animals for PrP3F4]). (E) Mean quantal parameters quantal size q. PrPP101L and PrPC NMJs. The estimations further current and fluctuation analysis showed roughly a 50% reduction revealed an increase in pvr at 2 and 3 mM [Ca2+]e in PrP3F4 but in those parameters following prion protein expression. (F) Double-logarithmic plot of the QC as a function of [Ca2+]e for all three genotypes as indicated. 0.9 + 0.org/ at University of South Dakota and School of Medicine on April 12. PrP3F4 versus PrPP101L: ∗∗∗ P . 23. 5) or direct eEJC and 630 + 104 (n ¼ 5) in controls to 288 + 91 (n ¼ 7) in PrPP101L mEJC measurements (Figs 2. Vol.7 + 0.2 Hz at different [Ca2+]e. 2015 Figure 6.05. 6E left.02 mM. 0. Note. EC50 ¼ 1. 171 + 5 and 194 + 15. PrPP101L (4. No. PrPC (2. Note data are from a different subset of experiments as in C (data denote mean + SEM).9. ANOVA with Tukey– Kramer post hoc test.05.3 and 2.3 + 0.7 + 0. 2014. (A) Group of eEJCs recorded from one NMJ at different indicated [Ca2+]e in mM. n ¼ 11 animals for PrPP101L. P ¼ 0. n ¼ 6 animals for PrPP101L. with n—number of NMJs indicated in bars [n ¼ 4 animals for control. 0. the analysis are consistent with the data from above experiments where cumu- showed that the number of release-ready vesicles decreased from lative eEJC amplitude measurements (Fig. vesicular release probabilities (pvr) at different [Ca2+]e and number of release-ready vesicles (N) estimated from fluctuation analysis in all three genotypes (∗ P .4 + 0. n ¼ 20– 27 NMJs [n ¼ 12 animals for control. Data denote mean + SD.23). n ¼ 5.0 + 0. PrPP101L tion of N from fluctuation analysis (600) in controls is in accord- showed a tendency towards enhanced pvr values (Fig. P ¼ 0. n ¼ 4 animals for PrP3F4]. ∗∗ P .001. 6E right. n ¼ 14 animals for PrPC].3∗ . respectively (Hill slope: PrP3F4 versus Ctrl: ∗ P . 0. ANOVA with Tukey– Kramer post hoc test. PrP3F4 causes a left-shifted [Ca2+]e—QC relationship but also and change in the slope of the fitted curves. Together. ANOVA). (C) Mean QC plotted versus different [Ca2+]e for the genotypes indicated and fitted with a Hill function according to QC([Ca2+]) ¼ QCmax[1+(EC50/[Ca2+])slope]21 which yielded QCmax ¼ 136 + 6. (D) Parabolic fits to the variance–mean relationships for the three genotypes indicated.oxfordjournals. The lines indicate regions used for analysis. ANOVA did not reveal any significance.3 + 0.01 mM and 0. thermore. Student’s t-test. Fig. and 246 + 30 (n ¼ 5) in PrP3F4 expressing larvae (Fig. 4.

Fig. Locomotive impairment is a commonly used generative signalling pathways are well-recognized and several diagnostic for characterization of models for neurodegeneration prion-related disease models in flies investigate ovine. expression of the PrP3F4/PrPP101L transgene induced the either functional or morphological alterations as developmental observed gain-of-function effect. all of which represent potential binding part- changes.62). phological studies (74) and our functional data support a role for logical roles are much less understood. A mutated PrPC (PrPP101L) exhibited diminished [Ca2+]e (Fig. Expression of either prion protein caused an increase in activity as detected in greater crawling distances Prion protein interaction with vesicle formation per 30 min (P . 6F). We have confirmed DISCUSSION presynaptic actions of PrP3F4 showing an increase in the size Our data demonstrate a novel physiological function of prion of synaptic vesicles. the possibility that prion diseases have loss-of- does not become the limiting factor. The rhythm (17) and neuronal excitability (67). The advantages of the Drosophila model to investigate neurode- tivities (63). The role for PrPC –laminin inter- action is consistent with scaffolding properties of PrPC and its role in regulation of signal transduction (70) with altered PrPC Prion protein mediates enhanced Drosophila larval signalling leading to synaptic dysfunction.4590 Human Molecular Genetics.36) or GSS-mediated (25. larvae were placed on a crawl. 6C) indicate a reduc. tial component of the active zone cytomatrix T-bar] per NMJ and most likely due to presynaptic mechanisms (32) supporting a the NMJ size by calculating the area of vGlut staining. 6).72) and a critical exam- a measure of low frequency muscle contraction would be the ination of this hypothesis depends on determining the elusive locomotor activity. 0. ter release at inhibitory synapses (14) and conversely. 2014. we have shown that PrP3F4 not only leads to larger vesicles but tion in Ca2+ co-operativity in PrP3F4 larvae but to corroborate also increases pvr with simultaneous reduction in the number this we analyzed the slopes of the linear regression lines to the of functional release sites equivalent to fewer ready-releasable double log QC – [Ca2+]e relationship at lower non-saturating vesicles.66).but also postsynaptic PrPC signalling precludes a precise determination of its function. Student’s (gain-of-function). The expectation would be that a stronger de. Figure 7 physiological role of endogenous PrPC in synaptic function. function components remains open (71. one could speculate that PrPC may play a role in .31. Various studies demonstrated a process (76). its physio. 23. As Downloaded from http://hmg. As PrPC interacts with synapsin (31) and its internalization is mission and mice lacking PrPC show reduced excitatory and mediated via clathrin-coated pits (75) in a dynamin-dependent inhibitory synaptic currents. circadian an altered Ca2+ co-operativity of release (58– 60). To test this behaviour. 8) relative to WT control larvae suggesting that the synaptic effects of prion Previous studies in mouse NMJs and hippocampal CA1 neurons protein expression also altered behavioural activities. P . Ca2+ release sites is due to functional rather than morphological channels or laminin.(28) and where foraging behaviours are affected (64) and this test was mouse-prion protein (26.org/ at University of South Dakota and School of Medicine on April 12. 2015 The detected reduction in the number of effective release sites the Drosophila genome does not contain an ortholog of mamma- as a consequence of prion protein expression could result from lian PrPC.68.30) neurode- employed to investigate locomotor activities of prion protein generation. Vol. shows that both parameters are unchanged between the three Recent evidence shows that PrPC has physiological roles and genotypes (three NMJs from three larvae per genotype. one would predict that deletion t-test. ANOVA) suggesting that prion protein expression does PrPC-KO/mutant models were observed (11–13). neuronal functions of PrPC. In the present study changes in the Hill slope shown above (Fig.32) consist- ent with PrPC expression at presynaptic terminals (73). ners for PrPC (13. PrPC is morphological changes at the NMJ are associated with various widely expressed and a loss-of-function would be observed in signalling pathways (61. The data show that the Ca2+ co-operativity phenotypes illustrating a dysfunctional signalling. Fig. 17 The enhanced QC seen in PrP3F4 expressing larvae can also be positive correlation between prion protein expression and synap- attributable to a change in Ca2+ dependence of release so we tic transmission (14. expressing larvae. neurite outgrowth (65.05.18. 0. was reduced following PrP3F4 expression (P . studies suggest that prion protein is required for synaptic trans. 0. Fig. Initial data from mouse PrPC in vesicular homeostasis. suggested that PrPC potentiates synaptic release (18. To distinguish between these two PrPC-KO mutants. Although many studies have reported and PrPC has been proposed at mammalian NMJs based on mor- neurodegenerative effects of misfolded prion protein.oxfordjournals. hence loss-of-function effects due to conversion into PrPSC or in 0. the postsynaptic muscle as long as the reduction in pool sizes However. pre. In fact. addition of lyzed the number of NC82 puncta [Bruchpilot (Brp)—an essen. metabotropic glutamate receptors. No. PrPC can inter- not induce any morphological changes and the reduction in act with synapsin Ib. 6) but not versus PrPP101L (P . In mammalian tissue. conversely. However. An interaction between synaptic vesicles protein at the synapse. This current study took advantage polarisation leads to a stronger contraction and further locomotor of the Drosophila model by using the UAS/Gal4 bipartite ex- distances.32) suggesting that PrPC is involved in determined whether the elevated transmitter release is due to neurotransmission (16. PrPC induces a potentiation of end-plate currents at mouse NMJs.69). On the behavioural level. locomotor activities The neurotoxic gain-of-function during prion disease is The outcome of the changes in synaptic transmission induced by believed to be due to accumulation of PrPSC but little evidence prion protein expression would be a stronger depolarization of is available that loss of PrPC plays a role during the pathogenesis. pression system with pan-neuronal PrP3F4/PrPP101L expression. of prion protein signalling would induce the opposite effect.05. Student’s As PrPC expression causes an increase in vesicle diameter t-test.001. ANOVA.05.27). PrPC-KO mice show reduced transmit- possibilities we performed confocal imaging of NMJs and ana. ing device allowing on-line monitoring of individual larval ac.

org/ at University of South Dakota and School of Medicine on April 12. Furthermore. RIM-binding protein family Importantly.oxfordjournals. release processes at the synapse and how a consequent conver. The number of NC82 puncta and vGlut area was calculated using Volocity software. 2015 Figure 7. P . vesicles (55. Vol. allows for the variability of reported numbers of AZs.56). . data denote mean + SEM. NMJ morphology is not affected by prion proteins. pool size The number of release sites determined by fluctuation analysis Our findings that PrP3F4 expression leads to an increase in pvr and (600) and postsynaptic current analysis (380) in our study is decrease in the size of the functional readily releasable pool sug. vesicle replenishment and release. a mutation in PrPC leads to impaired mem- sion of PrPC into PrPSC could induce synaptic dysfunction. In this context. PrPP101L and PrP3F4 expressing NMJs co-stained with anti-vGlut C-terminus and NC82 (anti-Brp) (Muscle 6). This interaction (78.05. similar to the number of AZs reported previously using serial EM gests further actions of PrPC in regulating synaptic transmission. (A) Confocal images were taken from control.77).49. images (500) (57). brane delivery of the a2d-1 subunit of VGCC in cerebellar granule neurons (13) suggesting that PrPC is required for intact synaptic Ca2+ signalling which may explain our results with Prion protein modulates release probability and vesicle respect to the increased pvr. endocytosis. or by differential and following synaptic strengthening at Drosophila NMJs coupling of voltage-gated Ca2+ channels (VGCC) with synaptic (42). (B) Mean data for counted NC82 puncta and vGlut area for the three genotypes indicated (n ¼ 3 NMJs from three animals for each genotype. Human Molecular Genetics. current analysis (380) (42) or fluctuation Changes in pvr could be caused by alterations in Ca2+ channel analysis (500) (39). ANOVA with Tukey– Kramer post hoc test). The fact that one AZ harbours not only one density at release sites which occurs in mutants of Rab3 and but two release-ready vesicles as shown at mouse NMJs (80) Rab3-interacting molecule (RIM) (39.79) illustrating an important connection between the would offer a new functional explanation of how PrPC modulates architecture of AZs and Ca2+-mediated vesicle release. No. 23. in our study PrP3F4 expression caused a reduction members and the cytomatrix-associated protein Brp are essential in the number of release-ready vesicles with simultaneously in binding Ca2+ channels and loss of RIM or mutations in Brp enhanced pvr as determined by independent methods demon- lead to a loss of Ca2+ channels (clustering) within the AZ strating a synaptic role of PrPC. 0. 2014. 17 4591 Downloaded from http://hmg.

ANOVA with increases in synaptic release.87) and this occurs in parallel with decreased Ca2+ MATERIALS AND METHODS co-operativity. is associated with faster and more Ca2+-efficient synaptic responses (56). Third instar larvae were put and release properties by PrPC increases synaptic strength. In re- on a moist.78). this lower co-operativity associated with higher pvr may also reduce the effects of residual Ca2+ build-up and thus Downloaded from http://hmg. members of the AZ architecture. Piccolo and RIMs (78. whereas PrP3F4 exhibits a reduced elav-Gal4 [c155] driver was obtained from the Bloomington . At the mouse calyx of Held. Tukey– Kramer post hoc test).001.82). the increase in co-operativity in PrPP101L could reflect a crucial function of PrPC in modulating Ca2+ signalling where this mutation shows a malfunctional phenotype. This connection may present a sig- organization of AZs is not static but undergoes plastic changes nalling pathway by which PrPC is required for intact Rab signal- (85) and PrPC could provide a potential candidate to interact ling. PrPC-mediated trafficking of VGCC sub- were monitored (A. Physiologi- cally. notion of an increased clustering of synaptic Ca2+ channels. Further experi- Is prion protein modulating calcium-mediated release? ments to elucidate the exact interaction of PrPC with the release machinery and synaptic ion channels.77.org/ at University of South Dakota and School of Medicine on April 12. Vol. will allow operativity as shown by a reduced Hill coefficient and slope of a better understanding of the physiological roles of PrPC the log – log QC – [Ca2+]e relationship (Fig. Functional differences between single release sites have been Bassoon. dependent release probabilities with concurrent reduction in the number of enlarged release-ready vesicles. 2015 prevent depletion of synaptic vesicles allowing a reduction of release-ready vesicles whilst preserving the synaptic strength as shown in our data. The PrPC interacting partner (90). proteins such as laminin b2 (scaffolding protein and PrPC binding partner). specifically mole- Our findings that PrP3F4 expression alters the Ca2+ co. Our data show similar co-operativity Flies were raised on standard maize media at 258C. Our data point towards a model where in prion disease resulting in synaptic dysfunction. In this context. 7). red dot—end point of tracking) units to the membrane which provides functional glutamatergic and data for each genotype were expressed as crawling distance (m) per 30 min (B) (n ¼ 11 animals for control. No. synaptic strength increases with devel- opment (86. Thus. food-free and temperature controlled (208C) surface. This identity depends on clustering and the number involved in synaptic transmission (22. and the facts that and activation of VGCC as shown at the calyx of Held (83. Data denote mean + SEM (∗∗∗ P . Our data provide new evidence for a functional role of Figure 8. Our morphological studies where prion protein signalling may lead to an optimization and indicate that PrP3F4 modulates the functionality rather than the increased efficiency of release. 0. 2014. n ¼ 17 animals for PrPP101L. in particular. The molecular mechanisms of AZ organization are important determinants of synaptic function. 17 value of 2. Prion proteins induce greater locomotor activities. Larval tracks lation to our findings. We propose a model.84) Rab3a activity is compromised in CJD (89) and Rab7a is a or the structure and architecture of the cytomatrix (57. based on the fact that PrPC interacts with several pro- teins involved in synaptic release (31. the prion protein expression results in fewer release sites with complex relationship between synaptic proteins and PrPC. 6) supports the signalling.88) are reported at Drosophila NMJs indicating a specific and heteroge- essential components. it is worth highlighting neous identity of each release site which determines its pvr the functions of Rab GTPases and RIM. In contrast. leading to higher Ca2+- number of AZs (Fig. may play a crucial role in PrPC-mediated animals for PrP3F4]). blue dot—starting point. thereby modulating neuronal transmission. including VGCC (13). The values in controls (3. The classical Ca2+ co-operativity usually refers to the co-operative interaction of 3 – 4 Ca2+ ions with the Drosophila stocks release machinery (58). which will subsequently be diminished with this structure. in a dynamic fashion. Larval locomotor PrPC in synaptic transmission where a modulation of vesicles behaviour was recorded over a period of 30 min. which are crucially (81.76) and a range of ion chan- nels.oxfordjournals. A reduced co-operativity and Hill slope (as seen with PrP3F4 expression) and increased synaptic strength are asso- ciated with tightening of VGCC to release sites in developmen- tally older compared with higher co-operativities and loosely coupled VGCC in younger synapses (55) suggesting that lower co-operativity. CONCLUSION In summary. n ¼ 21 transmission (13). suggests a novel physiological role. with those remaining having greater pvr possibly due to tighter coup- our data pointing towards an interaction of PrPC with AZ func- ling of Ca2+ channel/release site complexes by interacting with tion. cules of the cytomatrix and Ca2+-mediated release.81). we investigated synaptic PrPC signalling in more detail.4. CAST/Erc2/ELKS2a.3). 23.4592 Human Molecular Genetics. facilitating release.

The number 70 NaCl. After perme- Ca2+ co-operativity was analyzed from synaptic current ampli. UK). The obtained for each cell and fitted with the parabolic function 3F4 epitope tag on Mo-PrP does not distort the normal topology Var(I) ¼ I 2/N + qI that was constraint to pass through the or functions of PrPC (91. 2015 potential postsynaptic effects.1 ms. decay and frequency values. Co-operativity coefficients were derived by fitting goat serum.org/ at University of South Dakota and School of Medicine on April 12.. son of the decay of the first and the last eEJC within a train did not tials were 260 mV. Larval locomotor assay All data were digitized at 10 kHz and for miniature record. anti-vGlut C-terminus (kind ficients estimated this way closely match coefficients derived by gift from Hermann Aberle. larval fillets were incubated at 48C overnight in solu- linear regression lines to log-transformed individual data tions of primary antibody. Average single line fit to the linear phase of the cumulative eEJC plot (the last eEJC amplitudes (stimulus: 0. referred number of independent release-ready vesicles. TEVC recordings using sharp-electrodes were made from ventral longitudinal Muscle 6 in abdominal segments 2 and 3 of third instar larvae using pClamp 10. 20 MgCl2.98 (Stoelting Co. an Axoclamp 900A amp. Nerve stimulation was performed amplitude at time zero by the mean mEJC amplitude recorded with an isolated stimulator (DS2A. Human Molecular Genetics.97). Cumulative postsynaptic current analysis lifier and Digidata 1440A (Molecular Devices. tal Studies Hybridoma Bank) 1:200 dilution and a-tubulin . 23.2 Hz). which was applied to the Drosoph- (10 –30 MV) were filled with 3 M KCl.2% bovine serum albumin and 2% normal ent larvae. pvr and q were calculated expression system to drive pan-neuronal expression excludes using the equations: q ¼ A/(1 + CV2) and pvr ¼ I(B/A)(1 + Downloaded from http://hmg.oxfordjournals. Paired-pulse in the same cell.96). Muscles were clamped to 260 mV and in the presence of 0. 5 KCl.2 Hz) at different intervals (20. Upon fitting the parabola. unless otherwise stated) (39). respectively. All lines were backcrossed to CV2) where CV2 is the coefficient of variation of the eEJC ampli- w1118 for at least six generations allowing the use of w1118 as con. Immunohistochemistry Third instar larvae were dissected in ice-cold phosphate buffered Ca21co-operativity saline (PBS) then fixed in 4% paraformaldehyde. 2014. tudes at a given [Ca2+]e concentration calculated as: CV2 ¼ trols (Figs 5 – 8. 5 HEPES and 0. Receptor desensitization was 258C as differences in recording temperature cause changes in not blocked as it did not affect eEJC amplitudes since a compari- glutamate receptor kinetics and amplitudes (93).92). A and B were obtained from the fitting parameters (53. Recording electrodes lative eEJC amplitudes (50).5 to 3 mM to give mean eEJC amplitudes (I). To calculate the QC in the train. stimuli (0. washed and placed onto a moist. ings. University of Du¨sseldorf) 1:2000 di- recording from several Ca2+ concentrations in single cells lution.95). food-free surface (with constant tudes. abilization with PBS-0. (UAS-Mo-PrP3F4 containing a proline-to-leucine substitution The mean eEJC is given by I ¼ Npvrq (52. Flies homozygous for the release probability and q the quantal size at each given UAS-PrP3F4 or UAS-P101LD constructs were crossed with [Ca2+]e. QC was estimated for each temperature of 208C (63)). 200 s recordings we analyzed to obtain mean mEJC ampli. 40. NMJs for a given genotype. The plots of the variance – mean were expressing either PrP3F4 or mutant prion protein PrPP101L. 17 4593 Stock Center (Indiana. The use of the UAS/Gal4 bipartite origin.94) with N being the at residue 101) on the second chromosome (P101LD. No. we used experiments were performed by applying five repetitive mean mEJC amplitudes measured before the train. gression lines were statistically compared. Co-operativity coef.1% Triton (PBS-T) and blocking with tudes recorded for each [Ca2+]e from several muscles of differ. pvr the vesicular to as P101LD PrPC [PrPP101L]) (25). Crawling activities were imaged over recording by calculating the ratio of eEJC amplitude/average 30 min using AnyMaze software v4. Materials were pur- chased from Sigma-Aldrich (UK) unless otherwise stated. Holding poten. of release-ready vesicles was obtained by back-extrapolating a ose. Estimated values were not corrected for variability in mEJC amplitude distribu- Electrophysiology tions or latency fluctuations (51. PBS-T containing 0.5 mM tetrodotoxin (Tocris. USA). The eEJC variance was calculated as previously flies homozygous for the elav-Gal4 driver to produce offspring described (52. Digitimer). NC82 (supernatant) anti-Brp (Bruchpilot.5– 3 CaCl2 (as specified). 100 and 200 ms) for each cell at each inter-spike-interval. 1 – 5 V) are based on the 200 ms of the train) to time zero. The number of release-ready mean peak eEJC amplitude in response to ten presynaptic vesicles was then obtained by dividing the cumulative eEJC stimuli (recorded at 0. mEJC and eEJC recordings were off-line low-pass filtered at 500 Hz and 1 kHz. (eEJCs standard deviation/mean amplitude)2. Developmen- giving coefficients of 3 – 4 in wild-type larvae. mEJCs were recorded ila NMJ previously (42). 500 ms) were responses were recorded from muscles with input resistances calculated as the difference between peak and baseline before ≥4 MV and resting potentials more negative than 260 mV at stimulus onset of a given eEJC. were used: AH6 anti-PrP (TSE Reagent Resource Centre. 115 sucrose. UK) 1:1000 dilution. chromosome (UAS-Mo-PrP3F4) or mutant mouse prion protein ranging from 0. 10 NaHCO3. 5 trehal. Age-matched third instar larvae (100– 120 h) were selected. The following antibody dilutions points for Ca2+ concentrations ≤1 mM and the slopes of the re. USA) in The apparent size of the RRP was probed by the method of cumu- hemolymph-like solution 3 (HL-3) (60). The extracellular HL-3 contained (in mM): reveal any significant difference in decay kinetics. Compton. Vol. USA) and mEJC amplitude followed by averaging recordings across all data were analyzed off-line. The transgenic flies contain a UAS Variance – mean analysis of eEJCs construct of either wild-type mouse prion protein on the third Approximately 40 eEJCs were elicited at different [Ca2+]e. All synaptic eEJC amplitudes during a stimulus train (50 Hz.

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