Stimulation of PrPC retrograde transport towards the Endoplasmic Reticulum increases accumulation of PrPSc in prion-infected cells

Prion diseases are fatal and transmissible neurodegenerative disorders characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC) denoted PrPSc. In order to identify intracellular organelles involved in PrPSc formation, we studied the role of the Ras-related GTP-binding proteins Rab4 and Rab6a in intracellular trafficking of the prion protein and production of PrPSc. When a dominant-negative Rab4 mutant or a constitutively active GTP-bound Rab6a protein was over-expressed in prion-infected neuroblastoma N2a cells, there was a marked increase of PrPSc formation. By immunofluorescence and cell fractionation studies we have shown that expression of Rab6a-GTP delocalizes PrP within intracellular compartments leading to an accumulation in the endoplasmic reticulum (ER). These results suggest that prion protein can be subjected to retrograde transport towards the ER and that this compartment may play a significant role in PrPSc conversion. Our results demonstrate that stimulation of retrograde transport by Rab6aQ72L in N2a cells induced an intracellular redistribution of the prion protein within the ER compartment, showing for the first time that PrP proteins can be subject to an ER retrograde transport. Furthermore, we have determined that accumulation of PrP molecules in the ER is accompanied by an increase of PrPSc formation. A: For each sucrose respectively refractometry BCA PDI used as a marker for the endoplasmic reticulum


Introduction
Prion diseases are fatal, neurodegenerative disorders in humans and animals, which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. These disorders exemplify a novel mechanism of biological information transfer characterized by the generation of an abnormally folded isoform of the cellular prion protein (PrP C ), denoted PrP Sc , which represents the major component of infectious prion particles (1). PrP C is a neuronal membrane glycoprotein whose function has not been fully characterized; PrP C is rich in αhelical regions but can be converted into PrP Sc , a proteaseresistant isoform rich in β-sheet content that accumulates in the brain of infected organisms (2).
It has been postulated that PrP Sc may physically interact with PrP C , acting as a 'nucleus' or template for the formation of new PrP Sc molecules. During biogenesis, PrP C transits through the secretory pathway and is modified both by glycosylation and addition of a C-terminal GPI anchor. It is delivered to the cell surface clustered in detergent insoluble domains (DIM) as has been observed for other GPI anchored proteins and signalling molecules (3,4). The PrP protein is then rapidly endocytosed and recycled back to the cell surface. During this process, PrP C is cleaved, probably in an acidic cellular compartment. In cellular models, the conversion of PrP C into PrP Sc is thought to occur after PrP C has reached the plasma membrane and has been reinternalized for degradation (5,6). The molecular mechanisms underlying the conversion reaction are enigmatic and the precise intracellular compartment where it occurs remains unknown.
Cells maintain several dynamic properties to internalize and secrete proteins in different ways.
The traffic of membranes between organelles occurs through vesicular or tubular intermediates that selectively convey proteins and lipids from one compartment to another under the control of specific proteins. In this context, small GTPases of the Rab family are believed to play a role of insuring accurate targeting or docking of transport vesicles with their acceptor membranes (7)(8)(9).
They operate as molecular switches by interacting with different sets of proteins in the inactive GDP or active GTP bound states (review in (10)). Individual members of the Rab family are uniquely localized in specific membrane compartments and control the direction and/or specificity of particular steps in intracellular protein trafficking ( Figure 1). The roles of specific Rab proteins have been partially defined by studies involving over-expression of mutant Rab proteins with defective guanine nucleotide binding properties.
The exact subcellular sites of the processing events involved in the genesis of PrP Sc remain to be determined. It is likely that many, if not all, of the steps in the intracellular trafficking of PrP are mediated by distinct members of the Rab family. Consequently, functional perturbation of Rab proteins known to be localized in specific subcellular compartments may help to define the routes by which PrP C is converted into PrP Sc . As a first step in testing this approach, we have examined the effects of dominant-negative or constitutively active mutations of Rab4 and Rab6a proteins on the formation of PrP Sc in a prion-infected neuroblastoma cell line.
Rab4 protein has been implicated in the regulation of membrane recycling from early endosomes to the recycling compartment or directly to the plasma membrane (11,12)and may be involved in the plasma membrane recycling of PrP C . Rab6a stimulates retrograde transport at the level of the Golgi and induces a progressive, microtubule-dependent redistribution of Golgi resident proteins to the endoplasmic reticulum (ER) (13,14). When the dominant-negative Rab4-GDP or the constitutively activated Rab6a-GTP mutants were over-expressed in prion infected N2a cells, we detected a marked increase in the conversion of PrP C to the pathogenic form PrP Sc . This effect was observed both with endogenous PrP C and with an exogenous 3F4-tagged-MoPrP (15). Immunofluorescence and cell fractionation studies demonstrated an accumulation of PrP in the endoplasmic reticulum in Rab6a-GTP expressing cells. Our data suggest that retro-transport of PrP C towards the ER increases the production of PrP Sc suggesting that this organelle plays an important role in PrP Sc formation -4 -

Reagents and antibodies
Cell culture reagents (Opti-MEM, L-Glutamine and Trypsin) were from Life Technologies Inc. and foetal calf serum from Bio-Whitaker. Secondary antibodies were from Jackson Immunoresearch (West Grove, PA, USA). All other reagents were from Sigma. Pri308,

Cell culture
N2a neuroblastoma cells stably transfected with wild-type MoPrP and infected with Chandler (N2aMoPrP-Ch) or 22L (N2aMoPrP-22L) prion strains have been described previously (16). These cells were routinely culture in Opti-MEM (Life Technologies Inc.) supplemented with 10% heat-inactivated foetal calf serum and penicillin-streptomycin and maintained at 37°C in 5% C0 2 in a biohazard Level 3 laboratory.

Transfection assays, proteinase K digestion and Western blotting
Rab4 wt , Rab4 S22N and Rab4 Q67L cDNAs were a kind gift from Dr. Mary McCaffrey.
Rab4 and Rab6a wt , Rab6a T27N and Rab6a Q72L cDNAs were subcloned in the eukaryotic vector pRK5myc downstream and in-frame with the Myc epitope MEQKLISEEDL and sequenced verified. N2aMoPrP-22L cells were transfected using Fugene 6 (Roche Molecular Biochemicals) or Lipofectamine (Life Technologies Inc.) according to the manufacturer's instructions. Four days after transfection, cells were washed in PBS and lysed for 20 minutes at 4°C in Triton/DOC lysis buffer (150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris pH 7.5 and protease inhibitors). After 3 min of centrifugation at 6,000 rpm, the supernatant was collected and assayed for total protein content with a BCA Protein Assay kit (Pierce). Protein concentration was adjusted with lysis buffer. For detection of PrP Sc , equivalent volumes of samples were digested with 16µg of proteinase K per mg of protein at 37°C for 30 min, and the digestion was stopped by incubation with Pefablock (1 mM) for 5 min on ice. The samples were centrifuged at 14,000 rpm for 45 min at 4°C and the pellets re-suspended in 25 µl of SDS loading buffer and boiled for 5 min. Proteins were separated by SDS-PAGE in 12% acrylamide gels and transferred onto Immobilon-P membrane (Millipore) in Towbin buffer containing 10% ethanol. The membrane was blocked with 5% non-fat dry milk in TBST (0.1% Tween 20, 100 mM NaCl, 10 mM Tris-HCl; pH 7.8) for 1 hr at room temperature. After an overnight incubation at 4°C with primary antibodies (anti-PrP SAF mix or SAF32 diluted 1/300 in 5% milk in TTBS, anti-PDI or anti-GS28 diluted 1/500 in TTBS) the membrane was washed 4 times in TTBS and incubated for 1hr with an anti-mouse horseradish peroxydase conjugate (Amersham Pharmacia Biotech) diluted 1/5000 in 5% milk in TTBS. The membrane was washed 4 times with TTBS and developed using enhanced chemiluminescence (ECL). Gel bands were quantified using Sigma Scan Image software.

Cell fractionation
To separate and enrich Golgi and ER membranes, N2aMoPrP-22L cells were homogenised by using a stainless steel ball-bearing homogeniser (HGM precision engineering, Heidelberg, Germany) in 0.25 M sucrose, 10 mM Tris-HCl pH 7.4, 1 mM magnesium acetate, and a protease inhibitor mixture in a final concentration of 1 volume of cell pellet per 5 volumes of homogenising medium. Sucrose gradients were performed as described in (17). 1 ml fractions were collected from the top of each gradient, assayed for protein content and methanolprecipitated. After centrifugation at 14,000 rpm for 20 min, the pellets were re-suspended in SDS loading buffer. PrP was assayed by running fractions on 12% SDS PAGE, transferring the proteins onto Immobilon membranes and performing Western blot analysis with Saf32 antibodies.

Immunofluorescence
N2aMoPrP-22L cells were fixed in 3.7% formaldehyde in PBS for 10

Effect of Rab proteins on endogenous PrP Sc formation
In order to interfere with PrP intracellular trafficking and processing, prion infected Combined with the data observed with endogenous PrP, these results strongly suggest that impairing the plasma membrane recycling of PrP (by over expressing Rab4 S22N ) and stimulating the retrograde transport of proteins towards the ER (by expressing Rab6a wt and Rab6a Q72L ) increased the formation of the PrP Sc conformation. These results are in agreement with previous findings showing that PrP Sc conversion occurs within intracellular compartments and suggest that PrP Sc formation may involve a retrograde transport of PrP molecules into the ER.

Rab6a Q72L expression induces accumulation of PrP in intracellular compartments.
Before proceeding with further analysis of PrP Sc formation in cells expressing mutant Rab proteins, an immunofluorescence study was conducted to determine the sub-cellular localisation of PrP in the presence of Rab proteins. Because the expression of Rab6a Q72L had the most dramatic effect on PrP C conversion into PrP Sc , we decided to focus our study on the role of this Rab6a mutant protein on PrP trafficking. N2a MoPrP-22L cells, that had been transfected with Myc-Rab6a Q72L , were analyzed by indirect immunofluorescence. The overall transfection efficiency for the Myc-Rab6a Q72L vector was estimated to be approximately 90%. As shown in Interestingly, we can also detect the presence of PrP in the nuclei of the cells, presumably in the nucleoli, as already described (18,19) The intracellular localization of PrP in Rab6a Q72L expressing cells was also determined by subcellular fractionation on sucrose density gradients. GS28 and PDI were used as markers for Golgi and ER fractions respectively. Mock-transfected or over-expressing Myc-Rab6a Q72L N2aMoPrP-22L cells were fractionated on an equilibrium sucrose gradient and the distribution of total PrP was determined by immunoblotting the subcellular fractions with anti-PrP antibodies. Figure 4 shows that PrP was most abundant in the ER-enriched fractions in Rab6a Q72L transfected cells.  Figure 5A). In mock or Rab6a Q72L transfected cells PrP C was completely digested with 10 or 16 µg/mg of proteinase K, suggesting that abnormally folded PrP proteins are efficiently degraded by the proteasome system. When the ERAD-proteasome pathway is inhibited, partially protease resistant PrP species can be detected (ALLN-treated cells). Equal amounts of PrP C were detected in the different samples before PK digestion ( Figure 5B). Figure 5C show the expression of Myc-Rab6a Q72L in the transfected cells. We conclude that the expression of Rab6a Q72L in prion-infected N2aMoPrP cells induced accumulation of PrP Sc and not PK resistant PrP molecules which failed to pass the ERAD proteasome system.

Discussion
A major conformational change is thought to be a key event in the conversion of PrP C into the abnormal PrP Sc isoform. To understand the molecular events involved in the pathogenesis of  (25,26).
In order to interfere with downstream steps of PrP trafficking, we studied the effect of wild-type or mutant Rab6a proteins on PrP conversion. Extensive studies have documented the localisation of Rab6a in the trans-Golgi cisternea and trans-Golgi network in mammalian cells. Highly overexpressed active forms of Rab6a (Q72L mutant or wild-type) stimulate retrograde transport from post-Golgi vesicles back to the trans-Golgi network, progressively relocating Golgi residents to the ER (13,14). Surprisingly, we found that over-expression of a wild-type or a constitutively active form of the Rab6a protein in prion-infected N2a cells resulted in a marked enhancement of PrP Sc production. Using immunofluorescence and subcellular fractionation techniques, we demonstrated that over-expression of Rab6a-GTP alters the sub-cellular localization of PrP, shifting the protein from the cell surface to intracellular compartments including the ER. Previous investigations on intracellular trafficking of mammalian PrP C in living cells have shown that the protein, once internalized, is targeted to membranous structures close to the nucleus (27) and reminiscent of the Golgi apparatus (28). Based on the model of Rab6a function in intra-Golgi trafficking, we propose that Rab6a-GTP stimulates retrograde transport of PrP molecules within the trans-Golgi compartment towards the ER. A possible physiological retro-transport of PrP C into the ER has been already suggested. Thus, post-Golgi PrP species are mis-located into the cytosol in cells following treatment with proteasome inhibitors (20). Likewise, the truncated protein PrP Q160Stop is prevented from leaving the Golgi apparatus and transported instead to the cytosol and nucleus (29). This retro-transport process is believed to occur only in the ER, suggesting that PrP C , like other proteins (30), can recycle into the ER presumably by a Rab6a controlled pathway. It will be important to understand the intracellular retrograde pathway of PrP C during endocytosis. Expression of Rab9-GDP and GTP bound mutant proteins did not influence PrP Sc formation (F.B., unpublished observation), arguing against a transport of PrP C from the late endosomes to the Golgi apparatus (31,32). Whether PrP C is transported to the Golgi directly from the early endosomes or the plasma membrane or downstream in the endocytic pathway from a lysosome remains to be determined.
It is interesting to speculate that PrP may be transported from the cell surface to the endoplasmic reticulum through a Rab6a regulated transport pathway. Our data are consistent with this possibility, but do not directly demonstrate it. This pathway has been previously postulated to be used for degradation of trans-membrane proteins that have been targeted for proteasomemediated degradation in the cytosol (33). Most proteins are subject to a stringent surveillance mechanism that causes retention of misfolded forms in the ER. Proteins retained in the ER are then eliminated by reverse translocation into the cytoplasm, followed by proteasome degradation.
Recent data demonstrate that several misfolded mutants of PrP C are partially retained in the endoplasmic reticulum (34) and, as well as wild-type PrP C , are degraded through the ERADproteasome pathway (20,21). Another hypothesis of Rab6a-induced PrP Sc formation could concern the accumulation of misfolded PrP C proteins in the ER due to Rab6a-GTP expression.
In non-infected N2a cells, proteasome inhibitors induce the appearance of detergent-insoluble and proteinase K resistant PrP C . Such a PrP C form could not be detected in non-infected N2a cells over-expressing Rab6a-GTP, eliminating this possibility. Therefore, the increase of detected PrP Sc in prion-infected cells expressing Rab6a wild-type or GTP bound proteins reflects a real PrP Sc conversion and not an accumulation of misfolded PrP C .
The increase of PrP Sc production described after stimulation of Rab6a-controled retrograde transport suggest either that a certain proportion of PrP Sc remains in the ER and can induce a conformational change of retro-transported PrP C , or that Rab6a-GTP stimulates PrP Sc retrograde transport towards the ER where it can seed the conversion of nascent PrP C molecules.
The initial conformational change leading to PrP Sc formation would then occur just after synthesis as already suggested following the observation that mutated PrP molecules become abnormal in the ER after their synthesis (24).
Gilch et al have shown recently that intracellular re-routing of prion protein prevents propagation of PrP Sc (35). In this study, the compound Suramin completely prevented the plasma membrane localization of PrP, which was re-routed directly to acidic compartments. In the light of these data and our results, it seems that plasma membrane localization of the prion protein, followed by endocytosis and retrograde transport towards the ER are necessary to induce its conversion. If any of these pathways is blocked, either with Suramin (35) In several studies determining the subcellular localization of mutant PrP molecules, ER retention seems to be a common feature in experimental familial prion diseases (28,(36)(37)(38). Whether ER located PrP molecules are involved in cellular neurodegeneration remains to be established.
Retention of abnormal proteins in the ER is known to trigger stress response pathways (39) and could explain the pathogenic effects of mutant PrPs. Furthermore, a newly described Ctm PrP, a trans-membrane form of PrP that is thought to be involved in prion-induced neurodegeneration, is completely retained in the ER (40). Multiple lines of evidence demonstrate that many pathogenic mutations interfere with normal PrP trafficking, and that an abnormal retention of PrP protein in the ER may contribute to prion-induced neurodegeneration. Our results shed some light on PrP Sc conversion mechanism by showing that PrP molecules can be subjected to retrograde transport towards the ER, and that stimulation of this translocation leads to an accumulation of PrP Sc , demonstrating for the first time that the endoplasmic reticulum may play a role in PrP Sc formation.