Neurobiology of Disease 67 (2014) 71–78

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Reversible symptoms and clearance of mutant prion protein in an inducible model of a genetic prion disease in Drosophila melanogaster A. Murali a, R.A. Maue b,c, P.J. Dolph a,⁎ a b c

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA

a r t i c l e

i n f o

Article history: Received 22 November 2013 Revised 18 February 2014 Accepted 20 March 2014 Available online 28 March 2014 Keywords: Inducible model of GSS P101L Reversibility Clearance of misfolded prion protein

a b s t r a c t Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt–Jakob disease, kuru and Gerstmann–Straüssler–Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein. © 2014 Elsevier Inc. All rights reserved.

Introduction Neurodegenerative diseases are devastating conditions that often occur late in life, affect specific neuronal populations and are ultimately fatal. With the identification of gene mutations that are linked to familial forms of some of these neurodegenerative diseases, it has now become possible to study these disorders in greater detail via transgenic animal models. A common feature of many neurodegenerative diseases is the accumulation of abnormal or aggregated proteins, in either extracellular or intracellular compartments (Gama Sosa et al., 2012). Drosophila melanogaster has been used successfully to model Alzheimer's disease (Iijima et al., 2004), Parkinson's disease (Feany and Bender, 2000) and several other neurodegenerative conditions. Important insights have been gained from these fly models (Bilen and Bonini, 2005; Muqit and Feany, 2002), which offer the advantages of superior genetics and short generation time. Gerstmann–Sträussler–Scheinker (GSS) syndrome is an inherited prion disease, that typically affects patients in mid-life and is characterized by progressive ataxia (Prusiner, 1997; Imran and Mahmood, 2011). The most common mutation associated with GSS patients is a proline to leucine conversion at codon 102 of ⁎ Corresponding author at: HB 6044 Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA. Tel.: +1 6036461092. E-mail address: [email protected] (P.J. Dolph). Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2014.03.013 0969-9961/© 2014 Elsevier Inc. All rights reserved.

the prion protein (Ghetti et al., 2003). Previously, we have shown that Drosophila expressing the murine P101L protein (the corresponding mutation to human P102L), in cholinergic neurons develop a neurological condition that is characterized by ataxia, spongiform brain degeneration as well as shortened lifespan (Gavin et al., 2006). These symptoms are seen when the P101L protein is expressed in cholinergic neurons but not in dopaminergic neurons (Gavin et al., 2006). Additionally, we showed that the P101L protein that accumulated in this system was protease sensitive but misfolded in nature, as marked by 15B3 positive staining, an indicator of misfolded prion protein (Gavin et al., 2006). This study showed that Drosophila can be used to successfully model GSS disease. However, the constitutive expression of mutant prion protein in this model is associated with increased lethality in late larval and pupal stages. In order to eliminate the possibility of developmental defects, it is important to restrict the expression of the misfolded P101L protein to adult flies. In addition, creating a system where we can manipulate the expression of mutant P101L protein allows us to explore interesting aspects of prion disease such as the potential for reversibility. Here we report the development of a temperature inducible system where wild type mouse prion protein (MoPrP) and mutant P101L protein accumulates in cholinergic neurons at 28 °C, but not at 18 °C. In order to allow for efficient comparison between the effects of MoPrP and P101L expression, we used site directed transgenesis to control for any variation caused due to insertion at different genomic regions. Expression of both MoPrP and P101L protein in cholinergic neurons of

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adult Drosophila causes locomotor and electrophysiological defects. The effect of wild type overexpression is consistent with previous studies in mice and Drosophila (Fernandez-Funez et al., 2009, 2010; Westaway et al., 1994). However, we are now able to show that in a transcriptionally controlled system, expressing P101L protein results in highly significant exacerbation of symptoms compared to wild type MoPrP. Furthermore, we show that accumulated mutant P101L protein can be cleared over time when the expression of the protein is terminated. Additionally, inhibition of the proteasome results in increased accumulation of P101L. This study provides valuable insights into the mechanisms of P101L mediated neuronal dysfunction and shows that clearance of the misfolded protein can result in a reversal of genetic prion disease symptoms. Materials and methods Fly stocks and plasmids The driver line abbreviated as Cha-GAL4 was described previously (Gavin et al., 2006). The w[*]; sna[Sco]/CyO; P{w[+mC] = tubP-GAL80 [ts]}7, w[1118]; P{w[+mC] = UAS-Prosbeta2[1]}1B stocks were obtained from the Bloomington stock center (Bloomington, IN) and was recombined with the Cha-GAL4 line to create a ChaGAL4GAL80ts driver line. Construction of DNA plasmids and transgenic fly lines To create site specific insertion lines, the P101L and MoPrP cDNA were subcloned into the pUASTattB vector. These plasmids were then purified using Qiagen Maxiprep kits (Hilden, Germany). Transgenic fly lines were generated off-site (Genetic Services Inc, Cambridge, MA). Both transgenes were inserted into the third chromosome attP2 site. Resultant transgenic flies were crossed with the ChaGAL4GAL80ts stock for expression in cholinergic neurons. All flies carrying the ChaGAL4GAL80ts construct were raised at 18 °C and then switched to 28 °C to induce GAL4 activity. Occasionally, flies that carried PrP constructs and Cha GAL4GAL80ts failed to express GAL4, as indicated by the absence of GFP expression after 2 days at 28 °C. These flies were eliminated from the study. Only flies that were homozygous for P101L or MoPrP were used for subsequent analyses. Inhibition of the proteasome was conducted in a background where the P101L transgene was not in the attP2 site. Electrophoresis and Western blotting Eight fly heads were homogenized in lysis buffer (50 mM Tris, pH 7.5, 10 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholate) with a 0.2 ml Kontes Micro glass tissue grinder. The homogenate was spun at 5000 rpm for 1 min to eliminate large fly exoskeletal parts. The supernatant was boiled with an equal amount of sample buffer for 5 min and then either loaded onto gels immediately or stored at −80 °C. The samples were fractionated on 15% polyacrylamide gels and transferred to charged PVDF membranes (Millipore, Bedford, MA) using a Semi Dry Blotting Unit (Fischer Biotech). Membranes were blocked with 2% Amersham ECL blocking reagent in PBST for 30 min at RT. The 27/33 antibody (generously provided by Dr. S. Supattapone, Dartmouth Medical School) was used at a 1:30,000 dilution in 5% milk. This antibody is known to detect both wild type PrP and the P101L variant. The membrane was then processed as described in Gavin et al. (2006).

PBSTx. Brains were then washed in PBSTx twice for 5 min each and blocked for 1 h in 3% Normal Goat Serum (in PBSTx). Brains were then incubated in 27/33 antibody overnight (1:1000). Samples were then washed 5 times, incubated in secondary antibody for 2 h and after 4 more washes in PBSTx were mounted in Prolong AntiFade (Sigma) for confocal microscopy. Imaging was performed using a Nikon A1RSi confocal microscope. P101L and MoPrP puncta associated with neurons were counted and their area measured using the area measurement tool of the Nikon S Elements Software. A minimum of 93 neurons were counted for these analyses. Climbing assays Climbing assays were performed as previously described in Gavin et al. (2006). Briefly, 25 to 30 flies were placed in a vial and the number of flies crossing a 4 cm mark at the end of 45 s was noted. Climbing assays were repeated three times for each genotype. Experiments were then triplicated using fresh sets of flies. Statistical analysis was performed using two-tailed Student t-test. Electrophysiological analyses Brains were prepared for electrophysiology as described previously in Gu and O'Dowd (2006). Briefly, whole brains were dissected in recording saline (in mM: 101 NaCl, 4 MgCl2, 1 CaCl2, 3 KCl, 5 glucose, 20.7 NaHCO3, 1.25 NaH2PO4, pH 7.2) containing papain (20 U/ml). Once the connective tissue was gently removed, the brain was given a quick wash in saline without papain and immediately transferred into the recording chamber. Brains were held in place as previously described in (Gu and O'Dowd, 2006) and were perfused with oxygenated saline (95% O2/5% CO2) throughout the recording period. Recordings were made at room temperature, and for a given brain, recordings were made from no more than 2 neurons before another brain was chosen. The GFP-positive cholinergic neurons in the preparation were visualized using an upright fixed stage Zeiss Axioskope microscope (Germany) equipped with a 40 × water immersion objective (Zeiss Achroplan, numerical aperture 0.75), Hoffmann modulation contrast optics, and a 41018 EGFP fluorescence filter. Spontaneous action potentials were recorded in the on-cell recording configuration after giga-Ω seals were formed with electrodes (9-15 MΩ) filled with recording saline. All recordings were acquired with an EPC9 amplifier containing an ITC-16 AD/DA interface (HEKA Instruments, Bellmore, NT, USA), a Dell XPS computer, and Pulse 8.8 software (HEKA). The signals acquired during the continuous 5 minute recording periods were filtered at 10 kHz, digitized at 100 μs per point, and stored for later analysis offline. Action potential frequency was determined using Matlab 7.0 software (MathWorks Inc., MA, U.S.A). Stability of the firing rate during the course of a recording was confirmed by evaluating the number and time course of action potentials in sequential 1 minute segments of the 5 minute recordings. Events were included in the statistical analyses only if they exhibited the characteristic time course of action potentials. For experiments involving tetrodotoxin (TTX), brains were isolated, transferred to the recording chamber, and recordings made from a cholinergic neuron for 2 1/2 min. Following this, the recording chamber was perfused with saline containing 1 mM TTX for either 1–2 min or 8–10 min. Recordings were then made for another 2 1/2 min before the chamber was then re-perfused with the TTX-free saline solution for 5– 8 min and a final recording was made. Results

Immunostaining of Drosophila brains Immunostaining of whole Drosophila brains was performed using a modified protocol (Wu and Luo, 2006). Briefly, Drosophila heads were fixed for 3 h in 4% paraformaldehyde in PBS at 4 °C. Heads were then washed in PBSTx (2% Triton X-100 in PBS) and dissected in ice cold

Temperature induced expression of mutant (P101L) prion protein in adult Drosophila results in locomotor and electrophysiological defects Previously, our lab successfully created a Drosophila model of Gerstmann–Sträussler–Scheinker (GSS) disease. We overexpressed a

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mutant (P101L) form of the prion protein in cholinergic neurons (Gavin et al., 2006) using the UAS-GAL4 system (Brand and Perrimon, 1993). Transgenic animals that accumulated mutant P101L protein showed locomotor defects, age dependent spongiform neurodegeneration and increased mortality compared with non-transgenic controls (Gavin et al., 2006). P101L protein that accumulated in this system was protease sensitive but reacted to 15B3 antibody, an indicator that the protein in the system is misfolded. This model established that expression of the mutant P101L mouse protein in cholinergic neurons results in neurodegenerative disease in flies. To bypass any developmental defects caused by expression of P101L, we temporally restricted expression of the P101L protein to adult flies. To achieve this, we introduced a temperature sensitive copy of the GAL80 protein (McGuire et al., 2004). In this system, the GAL80ts protein inhibits the action of the GAL4 transcription factor at its permissive temperature (18 °C) but not at a higher temperature (28 °C). We also expressed wild type mouse prion protein (MoPrP) in this system. In order to compare the effects of wild type MoPrP and P101L without the complications of chromosomal position effects, we inserted both transgenes, into the same genomic region within flies (Markstein et al., 2008). This system allows for comparable levels of expression from both transgenes, which was confirmed by measuring mRNA levels (Fig. 1A) and protein levels (Fig. 1B). When the newly generated flies were raised at 18 °C (i.e., the permissive temperature of GAL80ts), the GAL80 protein was able to block GAL4 mediated expression, as indicated by the lack of expression of prion protein. Newly eclosed flies that were switched to 28 °C, however, exhibited expression of P101L or MoPrP protein within 2 days (Fig. 1B) and continued to accumulate the prion protein throughout the observation period of 8 days (Figs. 1C, D). Adult flies that expressed P101L protein displayed locomotor defects, which were measured using a climbing assay. Only 24% of flies expressing the P101L protein passed the climbing assay after 2 days at 28 °C. Flies that overexpressed wild type prion protein also showed locomotor defects as seen in other systems (Fernandez-Funez et al., 2009; Fernandez-Funez et al., 2010; Westaway et al., 1994). However, flies that expressed wild type prion protein performed significantly better on the behavioral test at every time point measured (Fig. 1E). In comparison, flies that were incubated at 18 °C exhibited no obvious locomotor defects. Flies expressing GFP at 28 °C in this system were also free from locomotor defects, ruling out any abnormalities in climbing that were caused due to elevated temperatures.

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We hypothesized that prion proteins might cause motor neuron defects by affecting the electrical activity of cholinergic neurons, similar to that seen in a Drosophila model of spinal muscular atrophy (Imlach et al., 2012). To test this, we adapted a previously described (Gu and O'Dowd, 2006) approach that allowed us to use cell-attached patch clamp recordings to measure currents associated with spontaneous action potential activity occurring in GFP expressing cholinergic neurons in whole brains isolated from adult Drosophila (Supplementary Fig. 1). Control cholinergic neurons expressing GFP and neurons expressing P101L displayed action potential-associated currents that were brief in duration (Figs. 2A, B), and could be reversibly inhibited by tetrodotoxin (TTX), a potent inhibitor of voltage-gated sodium channels (Supplementary Fig. 1). The level of spontaneous activity in the cholinergic neurons expressing the mutant P101L protein was more than 82% lower than in the control neurons (Fig. 2C). This significant (p b 0.0001) decrease in action potential frequency indicates that expression of P101L causes electrophysiological defects in the cholinergic neurons. Wild type MoPrP protein expression also resulted in dampening of spontaneous action potentials but similar to the behavioral analysis, expression of P101L resulted in significantly more serious defects. Given the severe effects of the disease relevant mutant form of the prion protein, we chose to focus our subsequent efforts on the P101L mutant. Locomotor and electrophysiological defects are reversed when expression of the prion protein is halted Our inducible model allows us to “pulse” flies at 28 °C to induce the expression of WT MoPrP or P101L protein and subsequently “chase” the effects by shifting the animals to 18 °C to block any further expression. We evaluated the locomotor behavior and electrophysiological responses of flies following this treatment regimen. As expected, flies that expressed P101L protein displayed locomotor abnormalities. Surprisingly, we found that these flies were able to recover near normal locomotor activity over time, when the expression of the protein was stopped. Of the flies that expressed P101L protein for 2 days and were then incubated at 18 °C for one day, only 26.8% passed the climbing test. After 10 days of recovery at 18 °C, however, there was a significant (p b 0.0001) improvement in the locomotive behavior of the flies, with 80.7% of flies passing the test (Fig. 3A). We found a similar trend in flies that accumulated P101L protein for 3 or 4 days with significant improvement (p b 0.001, p b 0.0001) in behavior when the expression of the protein was stopped and flies were allowed

Fig. 1. Temperature induced expression of MoPrP or P101L prion protein. (A) mRNA levels of wild type MoPrP and P101L protein are comparable after induction for 1 day or 2 days at 28 °C (B) Protein levels of MoPrP and P101L after 2 days at 28 °C (C, D) Western blot analysis shows expression of the MoPrP (C) or P101L (D) transgenes when flies are incubated at 18 °C (left) or 28 °C (right) for the indicated number of days. Accumulation of MoPrP or P101L protein occurs only at 28 °C. Tubulin was used as loading control. (E) Flies expressing P101L protein show severe climbing defects compared to MoPrP and non-transgenic controls incubated at 28 °C and control flies incubated at 18 °C where no expression of the P101L protein is expected. Each point on the graph represents flies that were able to climb above the 4 cm mark on a vial. Error bars represent S.E.M. ** indicates p = 0.01, and *** indicates p b 0.002.

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Fig. 2. Expression of P101L decreases action potential frequency of cholinergic neurons. (A) Representative on-cell recording from a cholinergic neuron, with a magnified and expanded segment of the recording shown below. For display purposes, inward currents during the action potential in the cell are represented by upward deflections of the current recording from the patch of membrane. (B) Representative cell-attached recording from a cholinergic neuron expressing GFP (control) at 28 °C. (C, D) Representative cell-attached recording from a cholinergic neuron expressing the P101L protein and MoPrP. In A–D, asterisks mark action potential events. (D). Quantification of action potential frequency when P101L, MoPrP or GFP is expressed for 3 days (induction for 3 days) or when the expression is followed by 10 days at 18 °C (n N 12 cells for all groups). Paired Student t test was used for statistical analysis. Asterisks indicate p b 0.0001.

to recover at 18 °C for 10 days (Figs. 3B, C). We observed that flies that expressed wild type mouse prion protein showed similar recovery in locomotor behavior (Figs. 3A–C). Similarly, when we analyzed electrophysiological responses, we found that the activity of cholinergic neurons that accumulated WT MoPrP or P101L protein for 3 days increased significantly (p b 0.001 for MoPrP and p = 0.001 for P101L) following a 10 day recovery period (Fig. 3D). In contrast, flies expressing WT MoPrP or P101L for 3 days had significantly dampened responses. This recovery of behavior is remarkable, and suggests that expression of prion protein does not cause immediate neuronal cell death but instead affects neuronal function early in the disease process. Previous studies have shown reversal of disease symptoms in an infectious model of prion disease (Mallucci et al., 2003; Mallucci et al., 2007; White et al., 2008). Here, we report for the

first time reversal in locomotor and electrophysiological dysfunction in a genetic model of prion disease. Wild type and mutant P101L prion proteins accumulate as large puncta on cholinergic neurons and the size of these puncta decrease during recovery To evaluate the cellular localization of the prion protein in the cholinergic neurons and to follow the localization of the protein during a “pulse chase” experiments, we used immunofluorescence analysis. Cholinergic neurons were identified by cytoplasmic expression of GFP (Fig. 4). We observed that prion protein was expressed on the surface of these neurons, which is the expected expression pattern for a glycophosphatidylinositol (GPI) anchored protein. Plasma membrane localization was confirmed by co-expression with a membrane specific

Fig. 3. Reversals of locomotor and electrophysiological defects occur when expression of wild type MoPrP or P101L is stopped at 18 °C. Flies were incubated at 28 °C for 2 days (A), 3 days (B), or 4 days (C), to induce expression of wild type MoPrP (dashed line) or P101L (solid line). Shifting flies to 18 °C results in recovery of locomotor behavior over time. By 10 days at 18 °C, significant (p N 0.001) improvement in behavior is observed in every time point. Expression of P101L appears to lead to more severe defects compared to WT MoPrP expression (D) Frequency of action potentials is significantly (p N 0.001, Student t-test) improved in flies shifted to 18 °C after a 3 day induction at 28 °C compared to flies expressing prion proteins for 3 days at 28 °C or temperature controlled flies expressing GFP in an inducible manner.

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these puncta decreases significantly (average size of P101L puncta: 0.12 ± 0.063 μm2, p value b 0.0001) after the expression of the protein is stopped for ten days indicating dispersal or clearance of the puncta (Figs. 5A, B). Of note, the size of the mutant P101L protein was significantly larger (p b 0.0001) than MoPrP puncta (average size at 1 day at 18 °C: 0.323 ± 0.015 μm2 and 0.08 ± 0.008 μm2 after 10 days at 18 °C). Accumulated prion protein is cleared from Drosophila neurons

Fig. 4. P101L is localized to large puncta on cholinergic neurons cell surface P101L or MoPrP (red) punctate staining pattern with cytoplasmic GFP or membrane bound mCD8-GFP (green) shows a staining pattern consistent with a GPI anchored protein.

marker (mCD8-GFP, Fig. 4) in the cholinergic neurons. Next, we followed the staining pattern of the prion protein throughout the course of a pulse-chase experiment. We observed that initially, both WT MoPrP and P101L protein accumulate in large aggregates or puncta on the surface of cholinergic neurons after 3 days at 28 °C (average size of P101L puncta: 0.447 μm2 (mean) ± 0.256 (S.D)). However, the size of

We observed robust expression of MoPrP after 2, 3 or 4 days of induction at 28 °C (Figs. 6A, B, C). After 10 days of recovery at 18 °C, a significant amount of the MoPrP protein was cleared, as evidenced by Western blot analysis (Fig. 5). We have quantified the amount of prion protein remaining at the end of the recovery period (10 days at 18 °C) relative to the initial amount of accumulated protein at the 1 day time point. Surprisingly, we found that the mutant P101L protein is also cleared from flies (Fig. 6). The decreasing levels of the prion protein correlates with improvement of behavioral symptoms in flies (Figs. 3A, B, C), indicating that clearance of the prion protein is an important step in the recovery process. Our results suggest the presence of endogenous mechanisms within Drosophila that are capable of clearing accumulated mutant protein. One attractive candidate is the proteasomal system which has been shown to play a role in clearance of other mutant prion proteins (Jin et al., 2000), (Ma and Lindquist, 2001). Proteasomal function in Drosophila can be impaired by the GAL4 mediated expression of prosβ2, a transgene that contains a dominant negative mutation in the β2 subunit of the 20S proteasome (Saville and Belote, 1993; Neuburger et al., 2006). When the proteasome is disrupted using prosβ2, flies accumulate more P101L protein compared to control flies (Fig. 5D), suggesting a

Fig. 5. Prion protein puncta size decreases over the course of recovery (A) Immunostaining pattern over the course of a pulse chase experiment. Flies were incubated at 28 °C for 3 days and then switched to 18 °C. Fly brains were sampled every 3 days subsequent to the switch to the lower temperature. No change in the number of GFP positive neurons was noted. (D) Quantification of the average size of MoPrP and P101L puncta over the time course at 18 °C. Mean values ± SD are (in μm2) for MoPrP are: 0.32 ± 0.01, 0.12 ± 0.004, 0.11 ± 0.009, 0.08 ± 0.008. Mean values for P101L are 0.44 ± 0.25 ± 0.25, 0.2 ± 0.09, 0.14 ± 0.07 and 0.12 ± 0.06. Asterisks indicates significant (p b 0.0001, Student t-test) differences between the 10 day and 1 day time points. Error bars represent standard error of mean. Scale bars represent 5 μm.

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Fig. 6. Clearance of wild type mouse prion protein after expression of the protein is stopped by incubation of flies at 18 °C. A–C demonstrate the clearance of MoPrP protein levels by the 10th day incubation at 18 °C compared to the initial amount of protein accumulated. Below each blot is the quantification of tubulin normalized-protein levels. Comparison between the first and tenth day time points are represented in the graph.

role for the proteasome in clearance of the P101L protein. Unfortunately, our model does not allow for direct testing of the proteasomal system involved in our “pulse chase” experiments since GAL4 is not active at the clearance temperature of 18 °C. However, our results show that the proteasome is involved in the degradation of the mutant protein as it accumulates within neurons, demonstrating that the proteasome is capable of eliminating misfolded P101L protein (Fig. 7). Discussion Gerstmann–Straüssler–Scheinker (GSS) syndrome is a genetic prion disease that is characterized by the onset and progression of ataxia (Ghetti et al., 2003). The most common mutation affecting patients with GSS disease is a proline to leucine substitution at codon 102

(P102L) (Ghetti et al., 2003). Here, we have successfully created a temperature inducible model of GSS disease by expressing the mouse homolog of human P102L protein. Previous studies have shown that overexpression of wild type mouse prion protein in both mouse and fly models also leads to the development of neurodegenerative illness (Chiesa et al., 2008; Fernandez-Funez et al., 2009; Fernandez-Funez et al., 2010; Westaway et al., 1994). In these cases, where over expression of wild type mouse prion protein causes neurological symptoms, mild reactivity of the prion protein to the 15B3 antibody, which recognizes misfolded prion protein, is observed (Chiesa et al., 2008; Gavin et al., 2006). Here, we have developed a system where we can directly compare wild type and mutant form of PrP since both transgenes are inserted into identical locations in the genome. We have shown that expression of wild type MoPrP also results in the development of

Fig. 7. Clearance of misfolded P101L protein occurs when expression of the protein is stopped and flies are allowed to recover at 18 °C. (A), (B) and (C) show that by 10 days at 18 °C, significant amounts of the P101L protein are no longer detected on a Western blot, indicating clearance of P101L. Graphs below the Western blots quantify the amount of P101L protein on the 10th day at 18 °C normalized to the amount of protein at the initial 1 day time point. (D) Accumulation of P101L protein is increased when the proteasome is disrupted by the introduction of prosβ2, a dominant, temperature sensitive proteasome subunit mutation.

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locomotor and electrophysiological defects. However, expression of P101L mutant protein results in a significantly worse outcome for flies. We have shown previously that flies expressing mouse P101L protein display many symptoms associated with prion disease, including locomotor defects and spongiform degeneration of fly brains (Gavin et al., 2006). We have also shown that the P101L accumulated in this system is protease sensitive, yet misfolded (Gavin et al., 2006). By creating a temperature inducible model, we are now able to regulate the expression of the P101L protein. We demonstrate that expression of P101L protein in adult flies causes severe locomotor defects. In addition, electrophysiological activity of individual cholinergic neurons expressing MoPrP or P101L protein is dampened. In each case, the locomotor and electrophysiological defects recorded was more severe in P101L expressing flies Expression of the prion protein in cholinergic neurons led to the formation of large punctate deposits on the cell surface, with P101L forming larger punctate structures compared to wild type MoPrP. Interestingly, we find that arresting the expression of both MoPrP and P101L results in recovery of normal behavior and electrophysiological responses over time. Importantly, we demonstrate that Drosophila are able to clear mutant prion protein from cholinergic neurons and show that clearance of the protein parallels the reversal of the behavioral and electrophysiological defects. We find that disrupting proteasomal systems leads to increased accumulation of P101L protein, indicating that that the proteasome is involved in the clearance of mutant protein. The reversibility of the disease implies that even at the stage of motor impairment, prion proteins cause neuronal dysfunction rather than cell death. It has been previously shown that the learning and memory defects in prion disease can be reversed when native prion protein is targeted either by deletion or via RNAi (Mallucci et al., 2003; Mallucci et al., 2007; White et al., 2008). Here, we provide the first evidence that the symptoms of a genetic prion disease can be reversed. We show that locomotor defects, which typify GSS disease and also represent a late stage symptom in non-genetic forms of prion disease, can be reversed via clearance of the misfolded protein. The previous studies mentioned, show that targeting cellular prion protein can prevent further accumulation of misfolded, disease causing forms of PrP in a scrapie infection model. We provide a complimentary approach by targeting mutant misfolded PrP itself. Upregulation of proteasomal degradation systems might prove to be an effective strategy in clearing misfolded prion protein in genetic prion diseases. Our electrophysiological data shows that in cholinergic neurons expressing wild type MoPrP or mutant P101L protein, there is a severe reduction in the level of spontaneous action potential activity. To our knowledge, this is the first instance of ex-vivo electrophysiological recordings targeting individual neurons expressing mutant prion proteins. Previous electrophysiological analyses of prion effects in mice have examined evoked excitatory postsynaptic field potentials (EPSPs) produced by large populations of neurons (Senatore et al., 2012) or have involved patch clamp recordings obtained from cultured cell lines (Solomon et al., 2010). We have extended these studies by using the patch clamp technique to record from single neurons in intact animals expressing prion protein. There are a variety of mechanisms that could underlie prion protein mediated inhibition of electrophysiological activity. Previous studies have shown synaptic localization of the prion protein (Herms et al., 1999) and it is conceivable that the aggregated proteins sterically hinder interactions at the synapse. Alternatively, the protein aggregates may interact with molecules at the cell surface preventing proper neurotransmission. Indeed, recent data show that the prion protein PG14 repeat, insertion mutant impairs glutamate release at the synapse by interacting with a voltage-gated calcium channel subunit (Senatore et al., 2012). Mutant prion proteins have also been shown to potentiate excitatory glutamate toxicity (Biasini et al., 2013). Alternatively, it has been proposed, that mutant prion proteins themselves form ion channels at the cell membrane (Solomon et al., 2012). The electrophysiological results we have

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obtained using this new model system, sets the stage for further detailed analysis of the electrical excitability and synaptic physiology of individual cholinergic neurons to determine how the P101L prion protein impairs neuronal function. In Drosophila, most excitatory neurons are cholinergic and motor neurons are glutamatergic (Baines, 2006; Salvaterra and Kitamoto, 2001). Yet in our system we see that expression of P101L protein as large puncta on cholinergic neurons leads to motor defects. In elegant studies done in the Drosophila larval motor system, impairing the activity of cholinergic sensory neurons and interneurons caused motor neuron dysfunction in a model of spinal muscular atrophy (Imlach et al., 2012). The authors showed that rescue of motor neuron defects could not be achieved by cell autonomous expression of the SMN protein in motor neurons. Instead, rescue was possible only when expression of the SMN protein was increased in cholinergic neurons. These experiments reveal that cholinergic neurons are an important component of an essential motor neuron circuit in Drosophila larvae, and that perturbing this circuit can lead to patterns of neurodegeneration. In the study described here, we provide evidence that in adult Drosophila, a similar motor circuit exists. Our results suggest that Drosophila have inherent mechanisms capable of degrading prion proteins, but this mechanism is overwhelmed when the protein expression is constant. The increased accumulation seen in the presence of the dominant negative proteasomal subunit suggests that the proteasomal system is involved in clearing P101L when it is being expressed and therefore, may also be involved in clearing accumulated protein after expression is terminated in our “pulse chase” system. P101L has been shown to be retained in ER compartments close to the nucleus in permeabilized cell culture systems (Ivanova et al., 2001). Previous reports have shown that other prion proteins harboring familial mutations are also retained within the secretory pathway (Ivanova et al., 2001; Ma and Lindquist, 2001; Singh et al., 1997). Therefore, the proteasome may be targeting prion protein that has accumulated within intracellular compartments. Previous studies have shown that other mutant forms of the prion protein can be cleared through the proteasome (Jin et al., 2000; Ma and Lindquist, 2001). Our results, in combination with these previous findings, indicate that enhancement of endogenous clearance mechanisms may have therapeutic potential. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.nbd.2014.03.013. Conflict of interest The authors declare no competing financial interests. Acknowledgments We wish to thank Surachai Supattapone and Vinoy Vijayan for the critical review of the manuscript, Elena Starostina for her help with immunostaining, Ann Lavanway for her assistance with confocal microscopy and Vidya Karunakaran for the help with quantifications of Western blots. The confocal microscope used for this study was supported in part by National Science Foundation Grant DBI-9970048 to Roger D. Sloboda. Thanks are also due to Diane O'Dowd, Leslie Henderson and their labs for technical assistance with electrophysiology. References Baines, R.A., 2006. Development of motoneuron electrical properties and motor output. Int. Rev. Neurobiol. 75, 91–103. Biasini, E., Unterberger, U., Solomon, I.H., Massignan, T., Senatore, A., Bian, H., Voigtlaender, T., Bowman, F.P., Bonetto, V., Chiesa, R., Luebke, J., Toselli, P., Harris, D.A., 2013. A mutant prion protein sensitizes neurons to glutamate-induced excitotoxicity. J. Neurosci. 33, 2408–2418. Bilen, J., Bonini, N.M., 2005. Drosophila as a model for human neurodegenerative disease. Annu. Rev. Genet. 39, 153–171. Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.

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Chiesa, R., Piccardo, P., Biasini, E., Ghetti, B., Harris, D.A., 2008. Aggregated, wild-type prion protein causes neurological dysfunction and synaptic abnormalities. J. Neurosci. 28 (49), 13258–13267. Feany, M.B., Bender, W.W., 2000. A Drosophila model of Parkinson's disease. Nature 404, 394–398. Fernandez-Funez, P., Casas-Tinto, S., Zhang, Y., Gomez-Velazquez, M., Morales-Garza, M.A., Cepeda-Nieto, A.C., Castilla, J., Soto, C., Rincon-Limas, D.E., 2009. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms. PLoS Genet. 5, e1000507. Fernandez-Funez, P., Zhang, Y., Casas-Tinto, S., Xiao, X., Zou, W.Q., Rincon-Limas, D.E., 2010. Sequence-dependent prion protein misfolding and neurotoxicity. J. Biol. Chem. 285, 36897–36908. Gama Sosa, M.A., De Gasperi, R., Elder, G.A., 2012. Modeling human neurodegenerative diseases in transgenic systems. Hum. Genet. 131, 535–563. Gavin, B.A., Dolph, M.J., Deleault, N.R., Geoghegan, J.C., Khurana, V., Feany, M.B., Dolph, P.J., Supattapone, S., 2006. Accelerated accumulation of misfolded prion protein and spongiform degeneration in a Drosophila model of Gerstmann–Straussler–Scheinker syndrome. J. Neurosci. 26, 12408–12414. Ghetti, B., Tagliavini, F., Takao, M., Bugiani, O., Piccardo, P., 2003. Hereditary prion protein amyloidoses. Clin. Lab. Med. 23, 65–85 (viii). Gu, H., O'Dowd, D.K., 2006. Cholinergic synaptic transmission in adult Drosophila Kenyon cells in situ. J. Neurosci. 26, 265–272. Herms, J., Tings, T., Gall, S., Madlung, A., Giese, A., Siebert, H., Schurmann, P., Windl, O., Brose, N., Kretzschmar, H., 1999. Evidence of presynaptic location and function of the prion protein. J. Neurosci. 19, 8866–8875. Iijima, K., Liu, H.P., Chiang, A.S., Hearn, S.A., Konsolaki, M., Zhong, Y., 2004. Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 101, 6623–6628. Imlach, W.L., Beck, E.S., Choi, B.J., Lotti, F., Pellizzoni, L., McCabe, B.D., 2012. SMN is required for sensory–motor circuit function in Drosophila. Cell 151, 427–439. Imran, M., Mahmood, S., 2011. An overview of human prion diseases. Virol. J. 8, 559 (422X-8-559). Ivanova, L., Barmada, S., Kummer, T., Harris, D.A., 2001. Mutant prion proteins are partially retained in the endoplasmic reticulum. J. Biol. Chem. 276, 42409–42421. Jin, T., Gu, Y., Zanusso, G., Sy, M., Kumar, A., Cohen, M., Gambetti, P., Singh, N., 2000. The chaperone protein BiP binds to a mutant prion protein and mediates its degradation by the proteasome. J. Biol. Chem. 275, 38699–38704. Ma, J., Lindquist, S., 2001. Wild-type PrP and a mutant associated with prion disease are subject to retrograde transport and proteasome degradation. Proc. Natl. Acad. Sci. U. S. A. 98, 14955–14960. Mallucci, G., Dickinson, A., Linehan, J., Klohn, P.C., Brandner, S., Collinge, J., 2003. Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis. Science 302, 871–874.

Mallucci, G.R., White, M.D., Farmer, M., Dickinson, A., Khatun, H., Powell, A.D., Brandner, S., Jefferys, J.G., Collinge, J., 2007. Targeting cellular prion protein reverses early cognitive deficits and neurophysiological dysfunction in prion-infected mice. Neuron 53, 325–335. Markstein, M., Pitsouli, C., Villalta, C., Celniker, S.E., Perrimon, N., 2008. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat. Genet. 40, 476–483. McGuire, S.E., Mao, Z., Davis, R.L., 2004. Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci. STKE 220, 16–25. Muqit, M.M., Feany, M.B., 2002. Modelling neurodegenerative diseases in Drosophila: a fruitful approach? Nat. Rev. Neurosci. 3, 237–243. Neuburger, P.J., Saville, K.J., Zeng, J., Smyth, K.A., Belote, J.M., 2006. A genetic suppressor of two dominant temperature-sensitive lethal proteasome mutants of Drosophila melanogaster is itself a mutated proteasome subunit gene. Genetics 173, 1377–1387. Prusiner, S.B., 1997. Prion diseases and the BSE crisis. Science 278, 245–251. Salvaterra, P.M., Kitamoto, T., 2001. Drosophila cholinergic neurons and processes visualized with Gal4/UAS-GFP. Brain Res. Gene Expr. Patterns 1, 73–82. Saville, K.J., Belote, J.M., 1993. Identification of an essential gene, l(3)73Ai, with a dominant temperature-sensitive lethal allele, encoding a Drosophila proteasome subunit. Proc. Natl. Acad. Sci. U. S. A. 90, 8842–8846. Senatore, A., Colleoni, S., Verderio, C., Restelli, E., Morini, R., Condliffe, S.B., Bertani, I., Mantovani, S., Canovi, M., Micotti, E., Forloni, G., Dolphin, A.C., Matteoli, M., Gobbi, M., Chiesa, R., 2012. Mutant PrP suppresses glutamatergic neurotransmission in cerebellar granule neurons by impairing membrane delivery of VGCC alpha(2)delta-1 subunit. Neuron 74, 300–313. Singh, N., Zanusso, G., Chen, S.G., Fujioka, H., Richardson, S., Gambetti, P., Petersen, R.B., 1997. Prion protein aggregation reverted by low temperature in transfected cells carrying a prion protein gene mutation. J. Biol. Chem. 272, 28461–28470. Solomon, I.H., Huettner, J.E., Harris, D.A., 2010. Neurotoxic mutants of the prion protein induce spontaneous ionic currents in cultured cells. J. Biol. Chem. 285, 26719–26726. Solomon, I.H., Biasini, E., Harris, D.A., 2012. Ion channels induced by the prion protein: mediators of neurotoxicity. Prion 6, 40–45. Westaway, D., DeArmond, S.J., Cayetano-Canlas, J., Groth, D., Foster, D., Yang, S.L., Torchia, M., Carlson, G.A., Prusiner, S.B., 1994. Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion proteins. Cell 76, 117–129. White, M.D., Farmer, M., Mirabile, I., Brandner, S., Collinge, J., Mallucci, G.R., 2008. Single treatment with RNAi against prion protein rescues early neuronal dysfunction and prolongs survival in mice with prion disease. Proc. Natl. Acad. Sci. U. S. A. 105, 10238–10243. Wu, J.S., Luo, L., 2006. A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat. Protoc. 1, 2110–2115.