The use of PrP transgenic Drosophila to replace and reduce vertebrate hosts in the bioassay of mammalian prion infectivity

Development of a Drosophila-based bioassay for mammalian prion infectivity

Overview. We have generated Drosophila transgenic for topological variants of mature length ovine PrP by pUAST / PhiC31-mediated site-directed mutagenesis. These fly lines, generated by Bestgene (California, USA), were transgenic for ovine PrP together with an N-terminal leader peptide and a C-terminal GPI signal sequence [PrP(GPI)] (Thackray et al., 2012c; Thackray et al., 2014a), or expressed ovine PrP without either an N-terminal leader peptide or a C-terminal GPI signal sequence [PrP(cyt)] (Thackray et al., 2014b). PCR and DNA sequencing was used in order to confirm that each PrP transgene was present as a single copy and located at the single 51D-site in the fly genome. We subsequently removed the red fluorescent protein (RFP) cassette located at the 51D site of the fly genome by Cre-mediated cleavage in each PrP fly line. Confocal microscopy of Drosophila S2 cells transiently transfected with the different pUAST-VRQ variants showed that PrP(GPI) and PrP(ΔGPI) entered the secretory pathway, whereas PrP(cyt) was restricted to the cytosol (Thackray et al., 2014a). Non-RFP UAS-PrP transgenic fly lines were subsequently crossed with GAL4-driver lines to allow expression of PrP in Drosophila.

PrP transgenic Drosophila were exposed to mammalian prions at the larval stage (Thackray et al., 2012b; Thackray et al., 2014a; Thackray et al., 2014b; Thackray et al., 2016). After hatching, Drosophila were transferred to prion-free culture tubes. At various time points during their adult lifespan, groups of Drosophila were analysed for locomotor ability, or euthanised, decapitated and homogenate prepared from the isolated fly heads (Thackray et al., 2012b; Thackray et al., 2014a; Thackray et al., 2014b; Thackray et al., 2016). These homogenates were used to seed in vitro PMCA reactions in order to reveal the presence of prion seeding activity (Thackray et al., 2014a); SDS/PAGE western blot detection of PrP^Sc; RNASeq-based transcriptome analysis (Bujdoso et al., 2015); or used in fly-to-fly or fly-to-mouse prion transmission studies (Thackray et al., 2016).

Fly stocks. The following fly lines were obtained from the Department of Genetics, University of Cambridge, UK.

Prion inoculation of Drosophila

Primary transmission of sheep scrapie (sheep-to-fly). Drosophila at the larval stage of development were exposed to brain homogenate of cerebral cortex tissue from a confirmed VRQ/VRQ PG127 (alternatively referred to as DAW or G₃₃₈) scrapie-positive sheep (SE1848/0005) (Thackray et al., 2008) or blood plasma from scrapie-positive sheep (Lacroux et al., 2012; Thackray et al., 2016). New Zealand-derived VRQ/VRQ scrapie-free brain tissue or blood plasma were used as control material. Two hundred and fifty microlitres of either 10% (v/v) blood plasma or 1% (w/v) of sheep brain homogenate, or a 1/10 dilution series (v/v) of these samples, prepared in PBS pH7.4, were added to the top of the cornmeal that contained third instar Drosophila larvae in 3-inch plastic vials. Following eclosion (i.e. hatching) flies were transferred to fresh non-treated vials.

Secondary transmission of sheep scrapie (fly-to-fly). Drosophila head homogenates were prepared from 30 day old flies that had been exposed at the larval stage to scrapie-positive or scrapie-negative sheep brain material. Two hundred and fifty microlitres of a 10^-1 (v/v) dilution of the original fly brain homogenate were added to the top of the cornmeal that contained third instar Drosophila larvae in 3-inch plastic vials. In all cases, flies were transferred to fresh, non-treated vials following eclosion.

Drosophila model characterisation and validation

Preparation of Drosophila head homogenate. Whole flies in an eppendorf tube were frozen in liquid nitrogen for 10 minutes and then vortexed for 2 minutes to cause decapitation. Individual fly heads were isolated and placed in clean eppendorf tubes using a fine paint brush. PBS pH 7.4 was added to give 1µL / head and homogenates were prepared by manual grinding of the fly heads with sterilised plastic pestles. For western blot analysis, fly head homogenate was mixed with an equal volume of 20% scrapie-free sheep brain homogenate prior to extraction and PK digestion as previously described (Lacroux et al., 2012) using monoclonal antibody Sha31 (Feraudet et al., 2005).

Protein misfolding cyclic amplification (PMCA). PMCA was carried out as previously described (Lacroux et al., 2012). The substrate consisted of 10% (w/v) ovine VRQ PrP (tg338) transgenic mouse brain homogenate in PBS pH 7.4, 0.1% Triton X-100 and 150 mM NaCl buffer (Lacroux et al., 2014). Five µL of fly head homogenate were mixed with 45µL of substrate in 0.2 mL thin wall PCR tubes. Sealed tubes were then placed in the horn of a Misonix 4000 sonicator for one round of 96 cycles. Each cycle consisted of a 10 second sonication step (70% of power) followed by a 14 minute and 50 second incubation step. Twenty µL of each reaction mix were subsequently treated with PK (4µ g of PK per mg of protein) at 37°C for 2 hours and the reaction stopped by adding Pefabloc (4mM final concentration). PK-resistant PrP was detected by western blot as previously described (Lacroux et al., 2012) using monoclonal antibody Sha31 (Feraudet et al., 2005).

Fly-to-mouse prion transmission. Fly-to-mouse prion transmission was carried out in ovine VRQ PrP (tg338) transgenic mice (Le Dur et al., 2005), which are highly efficient for the detection of ovine prion infectivity. All mouse bioassays were performed under licence number D-31-555-27, in compliance with institutional and national guidelines including ethical approval, and in accordance with the protection of animals used for scientific purposes under European Community Council Directive 2010/63/UE. Female tg338 mice (n=6) bred in-house aged 12 – 14 weeks were housed in a single cage with environmental enrichment and maintained under controlled conditions with respect to lighting, temperature, humidity and noise. Mice were injected intracerebrally with 20µL of diluted fly head homogenate (to give approximately 2 fly head equivalents per mouse) and monitored daily until the occurrence of clinical signs of mouse prion disease. Inoculated mice were euthanised when they started to show locomotor disorders and any impairment in their capacity to feed, or at a pre-defined end-point for the assay (>250 days) (Andreoletti et al., 2011). Brain tissue (cerebral cortex) was collected from euthanised mice and frozen for PrP^Sc analysis by Western blot (TeSeE, BioRad) or PET blot analysis (Andreoletti et al., 2011).

Negative geotaxis climbing assay

The locomotor ability of flies was assessed in a negative geotaxis climbing assay initiated with 45 (3 × n=15) age-matched, pre-mated female flies in each treatment group (Nichols et al., 2012; Thackray et al., 2014a; Thackray et al., 2014b). Drosophila were placed in adapted plastic 25mL pipettes that were used as vertical climbing columns and allowed to acclimatise for 30 minutes prior to assessment of their locomotor ability. Flies were tapped to the bottom of the pipette (using the same number and intensity of taps on each occasion) and then allowed to climb for 45 seconds. At the end of the climbing period the number of flies above the 25mL mark, the number below the 2mL mark and the number in between the 2mL and 25mL mark was recorded. This procedure was performed three times at each time point. The performance index (PI) was calculated for each group of 15 flies (average of 3 trials) using the formula: PI = 0.5 × (ntotal + ntop – nbottom)/ntotal where ntotal is the total number of flies, ntop is the total number of flies at the top, and nbottom is the total number of flies at the bottom. A PI value of 1 is recorded if all flies climb to the top of the tube whereas the value is 0 if no flies climb the tube past the 2mL mark. The mean PI ± SD at individual time points for each treatment group was plotted as a regression line.

Detailed methodology of the climbing assay is as follows:

Preparation of climbing assay pipettes

Plastic 25mL pipettes used in the climbing assay were prepared by taking a sharp saw blade and carefully cutting the top off the pipette. The cut edges were filed down in order to prevent damage to the Drosophila wings when the flies were added to, or removed from, the pipettes before or after the climbing assay was carried out. The tip of each pipette was sealed with a small piece of nescofilm wrapped securely around the point in order to prevent the escape of Drosophila during the assay. Clean cotton wool plugs were pushed into the top of each pipette to ensure a close fit so that the Drosophila could not climb out of the pipette once the assay had started.

Addition of flies to the climbing assay pipettes

At the start of each assay, the flies were counted in each set of fly vials dedicated to each treatment group in order to verify the number present (typically 3 vials, each containing 15 flies at the start of the experiment). The Drosophila from one vial were gently tipped into the top of a pipette using a dedicated plastic funnel for each treatment group. The cotton wool plug was securely fitted to stopper the top of the pipette as soon as the funnel was removed. The Drosophila were tapped to the bottom of the pipette, which was then laid horizontal and the flies allowed to acclimatise prior to the assay.

Acclimatisation of flies in the climbing assay pipettes

The pipettes that contained the Drosophila were placed horizontal at 25°C for 30 minutes in order to allow the flies to acclimatise. After the acclimatisation period the Drosophila were ready to start the climbing assay.

Pre-test climbing assay procedure

The tip of the climbing assay pipette was gently tapped a sufficient number of times on the bench to gather the flies together at the bottom of the apparatus, which was subsequently placed in a tube rack in an upright position at room temperature. The flies were allowed to climb for 45 seconds. There was no recording of data from this run as its purpose was to allow the flies to ‘practice’ climbing in the pipette that was held in an upright position.

Actual climbing assay procedure

Once the pre-test procedure had been completed, the climbing assay pipette was tapped gently on the bench (using the same number and intensity of taps as for the pre-test) and the apparatus was placed upright in the tube rack at room temperature. Drosophila were allowed to climb for 45 seconds. During the 45 seconds the number of flies to climb above the 2mL and 25mL marks were recorded. At the end of the 45 seconds, the number of flies above the 25mL mark, the number below the 2mL mark and the number in between the 2mL and 25mL mark was recorded. The whole climbing assay was repeated 2 more times to give a total of 3 readings per pipette.

End of climbing assay procedure

When all 3 climbing assay procedures had been performed, the Drosophila were gently tapped away from the cotton wool plug so it could be removed from the pipette without the loss of any flies. The Drosophila were then returned to fresh food vials using the dedicated plastic funnel for each group. Once the flies were back in fresh culture vials, the numbers of flies were counted and the number recorded on the lid to confirm that no flies had been lost during the assay or during the transfer to or from the pipettes. Climbing assay pipettes were checked to ensure no flies were stuck in the bottom of the pipette. The culture vials were returned to 25°C for routine fly maintenance.

PrP transgenic Drosophila to bioassay mammalian prion infectivity

Drosophila have proven to be a versatile experimental invertebrate host for use in the study of mammalian neurodegenerative diseases (Bilen & Bonini, 2005; Lu & Vogel, 2009). Several important features of Drosophila have aided this development. Firstly, Drosophila and mammals show conservation of basic components of the nervous system (Hirth & Reichert, 1999); Secondly, the genetics of Drosophila are well-defined, which allows the generation of transgenic flies with tissue-specific transgene expression. Third, the normal physiology and development of Drosophila is sufficiently well established to allow the use of behavioural assays that detect neurotoxicity in the living organism (Marsh & Thompson, 2006). Fourth, large numbers of Drosophila are readily generated in a short time and since this organism has a relatively short life span allows the rapid collection of, statistically robust data (Piper et al., 2005).

In order to develop an invertebrate-based bioassay for mammalian prion infectivity, we have generated ovine PrP transgenic Drosophila and have assessed the ability of these flies to detect ovine scrapie prions.

Generation of PrP transgenic Drosophila and prion inoculation

The Drosophila genome does not contain an orthologue of mammalian PrP and cellular expression of this protein is required for prion-induced neurotoxicity, which occurs during prion replication (Büeler et al., 1993; Mallucci et al., 2003). We exploited the successful application of PrP transgenesis to modify the susceptibility of a host for prion replication (Crozet et al., 2001; Thackray et al., 2012a; Vilotte et al., 2001) in order to explore Drosophila as a new animal model to assess mammalian prion infectivity.

Although PrP^C is primarily attached by a GPI anchor to the external side of the cell membrane, topological variants of the protein, including cytoplasmic and secreted forms, can arise during its biogenesis and metabolism (Borchelt et al., 1993; Chakrabarti et al., 2009; Hay et al., 1987; Hegde et al., 1998; Kim & Hegde, 2002; Stewart & Harris, 2003; Taylor et al., 2009). The role of these different forms of PrP in prion-mediated toxicity is not fully clarified. Accordingly, we generated Drosophila transgenic for the mature form of ovine PrP (amino acid residues 25 – 232) that was flanked by an N-terminal leader peptide and a C-terminal GPI signal peptide, which allowed expression of ovine PrP in the fly that was targeted to the plasma membrane, hereafter referred to as PrP(GPI) (Thackray et al., 2012c; Thackray et al., 2014a). In addition, we generated Drosophila transgenic for the mature form of ovine PrP that lacked the N-terminal leader peptide and C-terminal GPI signal peptide, which restricted PrP expression to the cytoplasm, hereafter referred to as PrP(cyt) (Thackray et al., 2014b). In order to generate Drosophila transgenic for these PrP variants we employed pUAST / PhiC31-mediated site-directed mutagenesis, whereby a single copy of the transgene of interest is delivered to the same landing-site in the fly genome in each respective fly line. Using this strategy, we demonstrated that different genotypes of ovine PrP protein could be successfully expressed in Drosophila. Expression of these ovine PrP variants in Drosophila had no adverse phenotypic effect upon the fly.

We subsequently tested the hypothesis that PrP transgenic Drosophila could bioassay exogenous ovine prions. To do so, Drosophila, at the larval stage, were exposed to sheep scrapie material known to contain prion infectivity as determined previously by transmission studies in mice (Thackray et al., 2008). Control inoculum consisted of known scrapie-free sheep brain homogenate. Drosophila were inoculated with scrapie-infected or scrapie-free sheep brain homogenate by addition of the material to larval feed. After hatching, flies were transferred to prion-free tubes and maintained for ≥40 days, during which time they were analysed for hallmark features of mammalian prion disease, namely the accumulation of infectious prions and evidence of a toxic phenotype.

Accumulation of prions in scrapie-exposed PrP transgenic Drosophila

We first investigated whether scrapie-exposed PrP transgenic Drosophila accumulated prions by measurement of prion seeding activity, a surrogate marker of PrP^Sc, using in vitro PMCA. Head homogenate prepared from scrapie-exposed, and control flies, was used as seed in PMCA together with brain homogenate from ovine PrP transgenic (tg338) mice as substrate. After amplification, the reaction mix was subjected to Proteinase K digest (PK) and the products analysed by western blot using an anti-PrP monoclonal antibody. Significantly, only reaction products of tubes seeded with head homogenate from scrapie-exposed PrP transgenic Drosophila showed the presence of PK-resistant PrP^Sc, which was good evidence for the presence of disease-associated PrP in the brains of these flies (Thackray et al., 2014a). This was supported by the presence of a potentially misfolded conformer of PrP evident by immunohistochemistry in scrapie-exposed ovine PrP transgenic Drosophila and insoluble PrP accumulation in these flies detected by conformation dependent immunoassay (Thackray et al., 2012b).

We next investigated whether bona fide infectious prions accumulated in scrapie-exposed Drosophila. This was addressed by fly-to-mouse transmission studies using ovine PrP transgenic (tg338) mice (Thackray et al., 2016). Remarkably, tg338 mice inoculated with head homogenate from scrapie-exposed PrP transgenic Drosophila developed mouse prion disease with 100% attack rate with a relatively rapid incubation time, indicative of a reasonably high level of prion infectivity in the fly head homogenate. The lack of detectable prion infectivity in head homogenate from scrapie-exposed control non-transgenic Drosophila argued against persistence of inoculum being responsible for the observed fly-to-mouse prion transmission (Thackray et al., 2016).

Prion-induced toxicity in scrapie-exposed PrP transgenic Drosophila

The presence of prion infectivity in scrapie-exposed PrP transgenic Drosophila was indicative of prion replication in these flies. Since prion-induced neurotoxicity occurs concomitantly with prion replication in mammalian hosts (Büeler et al., 1993; Mallucci et al., 2003), we investigated whether scrapie-exposed PrP transgenic Drosophila demonstrated a toxic phenotype. We assessed whether prion-exposed Drosophila showed any movement defects, since clinical signs of scrapie infection in sheep include locomotor defects, such as ataxia (Jeffrey & Gonzalez, 2007). To do so, we performed a negative geotaxis climbing assay (Nichols et al., 2012; Thackray et al., 2014a; Thackray et al., 2014b) using adult Drosophila exposed at the larval stage to ovine scrapie. After hatching, scrapie-exposed ovine PrP transgenic Drosophila showed an accelerated decline in locomotor activity. The severity of the locomotor defect increased as the flies aged, indicative of progressive illness. We also assessed whether scrapie-exposure affected the survival of PrP transgenic Drosophila since mammalian prion diseases are invariably fatal in affected individuals. Following exposure to scrapie material at the larval stage, adult PrP transgenic Drosophila showed a significantly enhanced mortality rate (Thackray et al., 2012b).

Collectively, these findings demonstrated that scrapie-exposed ovine PrP transgenic Drosophila accumulated prions that were transmissible to a mammalian host. Prion accumulation in the fly was associated with a progressive toxic phenotype evident as a locomotor defect. These hallmark features of mammalian prion disease in the fly were prion-mediated and PrP dependent since the effects were not observed in PrP transgenic Drosophila exposed to normal sheep brain material and were not displayed by scrapie-exposed flies that lacked PrP expression. These observations show that PrP transgenic Drosophila can be used to bioassay mammalian prion infectivity.

Sensitivity of PrP transgenic Drosophila to exogenous prions

In order to determine the sensitivity of the fly-based prion bioassay, ovine PrP transgenic Drosophila, at the larval stage, were exposed to a 1/10 (v/v) dilution series of scrapie-infected sheep brain homogenate. After hatching, the locomotor ability of adult prion-exposed Drosophila was assessed by a negative geotaxis climbing assay as the flies aged. We observed that the accelerated decline in locomotor ability displayed by adult PrP transgenic Drosophila diminished upon exposure to increasing dilution of scrapie-infected brain homogenate at the larval stage (Thackray et al., 2016). A statistically significant decline in locomotor ability was induced in PrP transgenic Drosophila by dilutions of scrapie-infected sheep brain homogenate in the range 10^-2 - 10^-10. For comparative purposes, we have used ovine PrP transgenic (tg338) mice to bioassay sheep scrapie-infected brain material. The tg338 mouse prion bioassay was able to detect sheep scrapie inoculum diluted to 10^-5, with the most dilute sample detected after a time course of ≈120 days in this mouse line (Andreoletti et al., 2011). These data showed that the Drosophila-based prion bioassay is of the order 10⁵-fold more sensitive than the tg338 mouse prion bioassay and can be completed in a significantly shorter time frame.

Detection of prion-infected blood by PrP transgenic Drosophila

The high level of sensitivity shown by ovine PrP transgenic Drosophila for ovine prions suggested the fly bioassay would be able to detect the low level of prion infectivity present in the blood of prion-diseased individuals. We tested this hypothesis by inoculating ovine PrP transgenic Drosophila with plasma samples from sheep experimentally infected with scrapie (Thackray et al., 2012b). We decided to bioassay plasma since this particular blood fraction has been reported to contain low levels of prion infectivity and has proven to be difficult to assess by conventional prion bioassay (Lacroux et al., 2012; Mathiason et al., 2010). We found that PrP transgenic Drosophila developed an accelerated decline in locomotor activity that became progressively reduced after exposure to more dilute samples of scrapie-infected plasma (Thackray et al., 2016). These observations were suggestive of titration of a particulate transmissible moiety in plasma obtained from scrapie infected sheep, a distinctive feature of the infectious scrapie agent (Stamp, 1962). The sheep plasma samples were known to contain scrapie prion infectivity as they had previously been transmitted to sheep and mice (Lacroux et al., 2012).

We also observed that plasma isolated from natural scrapie-infected sheep could induce a toxic phenotype in ovine PrP transgenic flies (Thackray et al., 2016). The response to natural scrapie plasma was evident with samples collected from asymptomatic scrapie-infected sheep aged ≥6 months of age and was more pronounced after exposure to plasma obtained during the clinical phase, which commenced around 20 months of age. Importantly, we determined through fly-to-fly transmission that the toxic fly phenotype induced by pre-clinical natural scrapie plasma was transmissible (Thackray et al., 2016). These observations showed that ovine PrP transgenic Drosophila could successfully bioassay a transmissible moiety in the blood of scrapie-infected sheep, which was detectable at an early pre-clinical time point.

Transfusion experiments in sheep show that whole blood from non-clinical ovine donors aged ≥3 months can be used to detect scrapie-infected animals (Lacroux et al., 2012). We consider that PrP transgenic Drosophila show a similar, if not greater, sensitivity than transfusion studies in the natural host since plasma from scrapie-affected sheep contains less prion infectivity than whole blood (Lacroux et al., 2012). Furthermore, the amount of time required to bioassay plasma in PrP transgenic Drosophila was significantly shorter than the case for transfusion studies in the natural host (McCutcheon et al., 2011).