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Prion protein facilitates synaptic vesicle release by enhancing release probability.

Robinson SW, Nugent ML, Dinsdale D, Steinert JR - Hum. Mol. Genet. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK.

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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. (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 α-tubulin. Note, PrPC can undergo glycosylation leading to multiple PrP bands (25,98) which are not detectable in larvae. n = 3 animals per lane. (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 μg/ml) showed no PK resistance. PK completely digested prion protein (∼25 kDa bands) at relatively low concentrations (∼1 μg/ml) and so is α-tubulin being digested. n = 5 larval heads per lane.
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DDU171F1: 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. (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 α-tubulin. Note, PrPC can undergo glycosylation leading to multiple PrP bands (25,98) which are not detectable in larvae. n = 3 animals per lane. (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 μg/ml) showed no PK resistance. PK completely digested prion protein (∼25 kDa bands) at relatively low concentrations (∼1 μg/ml) and so is α-tubulin being digested. n = 5 larval heads per lane.

Mentions: Expression of wild-type murine PrPC (PrP3F4) in Drosophila causes spongiform degeneration in adult fly brains (26) and importantly this degeneration is accelerated following expression of a mutated PrPC (P101L) [PrPP101L], a mutation which is linked to the human prion disease GSS. In initial experiments we aimed to validate expression of either PrP3F4 or the mutated prion protein (PrPP101L) in transgenic Drosophila larvae by performing immunohistochemistry (IHC) which confirmed strong and specific expression of either protein within all boutons of the NMJ and lack of expression in UAS controls [Fig. 1A, co-stained for vesicular glutamate transporter (vGlut)]. Western blot analysis further confirmed expression of either prion protein (Fig. 1B). Assessing expression levels of both prion proteins revealed no differences between both lines (prion protein/α-tubulin ratio: PrP3F4: 1.2 ± 0.4 (n = 10), PrPP101L: 1.0 ± 0.2 (n = 7), P = 0.35, Student's t-test). As PrPC expression induces a neurodegenerative phenotype in older adult flies (26,30) we next tested whether the expressed prion proteins were sensitive to proteinase digestion at these developmentally younger larval stages. In order to test this we employed the Proteinase K (PK)-digestion protocol. An increased resistance to PK digestion has been shown in older flies (30 days) expressing PrP3F4 where digestion occurs above ∼5–7 μg/ml (30,36) but it is unknown whether this resistance to PK digestion, an indicator of PrPSC misfolding, is evident in third instar larvae. Any digestion occurring below ∼5–7 μg/ml could suggest a lack of misfolding and that the putative function of these proteins may not be affected (12). Incubation of larval preparations with various concentrations of PK (Fig. 1C, 0–1 μg/ml) did not indicate any formation of PK-resistant prion in either PrP3F4 or PrPP101L transgenic lines as PrP3F4 and PrPP101L digestion starts at ∼1 μg/ml (so is α-tubulin being digested by this concentration of PK, Fig. 1C). Thus, protein misfolding and aggregation is unlikely to occur at this developmental stage (12). It is established that synaptic loss in murine prion disease models precedes degeneration of the cell soma (34) but it remains to be investigated what the underlying mechanisms for this dysfunction are and whether pre- or postsynaptic prion protein signalling is involved. The current study aims to investigate prion protein-mediated effects on presynaptic release mechanisms to elucidate its physiological roles.Figure 1.


Prion protein facilitates synaptic vesicle release by enhancing release probability.

Robinson SW, Nugent ML, Dinsdale D, Steinert JR - Hum. Mol. Genet. (2014)

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. (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 α-tubulin. Note, PrPC can undergo glycosylation leading to multiple PrP bands (25,98) which are not detectable in larvae. n = 3 animals per lane. (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 μg/ml) showed no PK resistance. PK completely digested prion protein (∼25 kDa bands) at relatively low concentrations (∼1 μg/ml) and so is α-tubulin being digested. n = 5 larval heads per lane.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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DDU171F1: 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. (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 α-tubulin. Note, PrPC can undergo glycosylation leading to multiple PrP bands (25,98) which are not detectable in larvae. n = 3 animals per lane. (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 μg/ml) showed no PK resistance. PK completely digested prion protein (∼25 kDa bands) at relatively low concentrations (∼1 μg/ml) and so is α-tubulin being digested. n = 5 larval heads per lane.
Mentions: Expression of wild-type murine PrPC (PrP3F4) in Drosophila causes spongiform degeneration in adult fly brains (26) and importantly this degeneration is accelerated following expression of a mutated PrPC (P101L) [PrPP101L], a mutation which is linked to the human prion disease GSS. In initial experiments we aimed to validate expression of either PrP3F4 or the mutated prion protein (PrPP101L) in transgenic Drosophila larvae by performing immunohistochemistry (IHC) which confirmed strong and specific expression of either protein within all boutons of the NMJ and lack of expression in UAS controls [Fig. 1A, co-stained for vesicular glutamate transporter (vGlut)]. Western blot analysis further confirmed expression of either prion protein (Fig. 1B). Assessing expression levels of both prion proteins revealed no differences between both lines (prion protein/α-tubulin ratio: PrP3F4: 1.2 ± 0.4 (n = 10), PrPP101L: 1.0 ± 0.2 (n = 7), P = 0.35, Student's t-test). As PrPC expression induces a neurodegenerative phenotype in older adult flies (26,30) we next tested whether the expressed prion proteins were sensitive to proteinase digestion at these developmentally younger larval stages. In order to test this we employed the Proteinase K (PK)-digestion protocol. An increased resistance to PK digestion has been shown in older flies (30 days) expressing PrP3F4 where digestion occurs above ∼5–7 μg/ml (30,36) but it is unknown whether this resistance to PK digestion, an indicator of PrPSC misfolding, is evident in third instar larvae. Any digestion occurring below ∼5–7 μg/ml could suggest a lack of misfolding and that the putative function of these proteins may not be affected (12). Incubation of larval preparations with various concentrations of PK (Fig. 1C, 0–1 μg/ml) did not indicate any formation of PK-resistant prion in either PrP3F4 or PrPP101L transgenic lines as PrP3F4 and PrPP101L digestion starts at ∼1 μg/ml (so is α-tubulin being digested by this concentration of PK, Fig. 1C). Thus, protein misfolding and aggregation is unlikely to occur at this developmental stage (12). It is established that synaptic loss in murine prion disease models precedes degeneration of the cell soma (34) but it remains to be investigated what the underlying mechanisms for this dysfunction are and whether pre- or postsynaptic prion protein signalling is involved. The current study aims to investigate prion protein-mediated effects on presynaptic release mechanisms to elucidate its physiological roles.Figure 1.

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.

View Article: PubMed Central - PubMed

Affiliation: MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, UK.

Show MeSH
Related in: MedlinePlus