Abstract
Abstract Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann-StrausslerScheinker disease (GSS), familial Creutzfeldt-Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrP s~) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP 202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP 2~ expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as 'curly' aggregates. In addition, PrP 2~ cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation.
Introduction
Transmissible Spongiform Encephalopathies or prion disorders are fatal neurodegenerative diseases of humans and animals that are infectious, familial, and sporadic in nature. The common underlying event in all prion disorders is a change in conformation of normal cell surface glycoprotein or prion protein (PrP c) from a mainly 0c-helical conformation to a [3-sheet rich isoform or PrP-scrapie (prpS~). PrP sc is the principal constituent of the 'prion infectious particle' and is also believed to be the main cause of neuronal death in prion disorders, regardless of their origin. Thus, exposure to PrP so, accidental or experimental, of otherwise healthy humans and animals leads to infectious disorders, while familial disorders result from point mutations in the prion protein gene (PRNP) , and sporadic disorders occur as a random event where PrP c undergoes a spontaneous change in conformation to PrP s~ (Aguzzi and Polymendiou 2004; Prusiner 1998) . Although a wealth of data implicate PrP s~ in prion disease associated neurotoxicity, other reports demonstrate prion specific neuropathology in the absence of detectable PrP s~, indicating the presence of additional or alternative pathways of neurotoxicity (Harris and True 2006; Lasmezas et al. 1997) . The identity of these neurotoxic signals, however, remains unclear.
In an attempt to identify additional or alternative factors contributing to prion disease associated neurotoxicity, we have investigated the less common familial prion disorders that are easier to model experimentally and have historically provided useful information about the more common but difficult to model sporadic disorders. Familial priori disorders comprise -15% of all cases, and are inherited as an autosomal dominant trait with almost -90% penetrance. These disorders exhibit a high degree of phenotypic variability and are classified as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker (GSS) syndrome, and fatal familial insomnia (FFI), based mainly on the phenotypic presentation. More than 30 different point mutations and octapeptide repeat insertions in PRNP segregate with these disorders, with little correlation between the mutation and the associated phenotype. Point mutations at codons P102L, P105L, A117V, G131V, Y145stop, F198S, D202N, Q217R, M232T, and insertions of octapeptide repeat sequences segregate with GSS, whereas mutations at codons D178N, V180N/M232R, T183A, E200K, R208H, V210I, M232R are associated with CJD (Aguzzi and Polymendiou 2004) . Although neuronal damage in most familial cases has been attributed to a spontaneous change in conformation of mutant PrP to PrP so, the correlation is less than perfect (Harris and True 2006; Lasmezas et al. 1997) . Recent evidence suggests that misfolding and consequent misprocessing of mutant PrP is an impo~t underlying metabolic defect, and may induce neurotoxicity by altering cellular function in the absence of detectable PrP sc (Ma and Lindquist 2001; Ma et al. 2002; Singh et al. 2005) .
To evaluate this possibility, we investigated the processing and metabolism of mutant PrP D202N (PrP 2~ associated with GSS in human neuroblastoma cells (M17). For comparison, cells expressing PrP 2~176 the most common form of familial CJD (Aguzzi and Polymendiou 2004; Capellari et al. 2000) , and prpC-expressing cells were evaluated in parallel. We report that majority of PrP 2~ synthesized in M17 cells does not acquire a mature conformation and is not expressed on the plasma membrane of transfected cells. Instead, greater than 60% of PrP 2~ is retained in the endoplasmic reticulum (ER) where it forms detergent insoluble aggregates. Intracellular accumulation of PrP 2~ causes upregulation of the ER chaperone calnexin. Surprisingly, aggregated PrP 2~ forms 'curly' or fibrillar deposits, a unique finding that has not been reported for any other mutant PrP form. In addition, cells expressing PrP 2~ (prp2~ are more sensitive to oxidative stress when exposed to H202, suggesting that improper folding of PrP 2~ interferes with normal PrP function as an anti-oxidant and increases the vulnerability of cells to oxidative damage (Brown et al. 2002 (Brown et al. , 1997 White et al. 1999) . Additionally, ER stress induced by intracellular PrP 2~ may further contribute to the above process (Hetz et al. 2005) , providing a mechanism by which mutant PrP D202N may induce neuronal death in the absence of detectable PrP so.
Materials and Methods
Cell Culture and Transfection Human neuroblastoma cells (M17) expressing PrP c and mutant PrP E200K (PrP 2~176 and D202N (PrP 2~ were generated and cultured as described (Gu and Singh 2004 
Cell Staining and Confocal Microscopy Cells expressing
PrP c, PrP 2~176 and PrP 2~ were immunostained and evaluated as described in previous reports (Mishra et al. 2002; Singh et al. 1997) . For co-immunostaining with anti-calnexin antibody, cells expressing PrP c and PrP 2~ were immunostained with 3F4-anti-mouseF1TC, and anti-Calnexin immune serum-anti-rabbit-TRITC. To examine the effect of free radicals, PrP c and PrP 2~ cells were exposed to 100 laM H202 for 30 min, followed by fixation, permeabilization, and staining with 3F4-anti-mouse-FITC and Hoescht. Stained coverslips were mounted on gel-mount (Biomeda Corp., Foster City, CA, USA) and observed using optical fluorescent microscope or a laser scanning confocal microscope.
Immunoblotting Cells expressing PrP c, PrP 2~176 or PrP 2~
were lysed in a non-ionic buffer containing a cocktail of protease inhibitors, and methanol-precipitated proteins were fractionated by SDS-PAGE and immunoblotted with 3F4 (1:40,000) or 8H4 (1:1000) essentially as described (Mishra et al. 2002) . To assay detergent insolubility, lysates of PrP c-and PrpZ~ were centrifuged at low speed to pellet nuclei and cell debris, and the pellet was kept aside as P1 fraction. Clarified supernatant or S1 fraction was further ultra-centrifuged at 100,000 g in a Beckman SW50 rotor for 1 h at 4~ to separate the highspeed pellet (P2) and supernatant ($2) fractions. All four fractions were analyzed by SDS-PAGE followed by western blotting and probed with anti-PrP antibody 3F4 (1:40,000).
Metabolic Labeling and Immunoprecipitation PrP c-and prp2~
were pre-incubated in methionine/eysteinefree medium containing 5% dialyzed serum for 1 h at 37~ and labeled with 50 p.Ci/mL of Tran-[35S]-label for 30 min or ovemight in the same medium. Subsequently, cells were either washed with cold phosphate-buffered saline (PBS) and lysed as above, or washed with Opti-MEM and chased as indicated. At the end of each time point, cells were washed with cold PBS, and clarified lysates were immunoprecipitated with 3F4 or 8H4. Bound proteins were eluted in boiling sample buffer (2% SDS, 10% glycerol, 5% [3-mercaptoethanol) and analyzed by SDS-PAGE-fluorography.
Results and Discussion
Mutant P r P 202N is Retained in the Endoplasmic Reticulum of Human Neuroblastoma Cells
To understand the metabolism of P r P 202N in transfected cells, steady state distribution of PrP c, PrP 2~176 and PrP 2~ was evaluated in human neuroblastoma cells expressing these proteins at comparable levels. PrP c represents the normal control, whereas PrP E200K is a well-studied mutant PrP form associated with familial CJD (Capellari et al. 2000) . Thus, cells plated overnight in I0 cm culture dishes at a confluency of 9x 106 were lysed in non-ionic detergents, and cellular proteins were precipitated with cold methanol followed by fractionation on SDS-PAGE and Western blotting. Immunoblotting with anti-PrP antibody 3F4 shows the expected glycoforms of PrP including the unglycosylated form of 27 kDa, mono-and di-glycosylated forms of 30 and 32 kDa, and the complex glycosylated form of 37 kDa. PrP 2~176 shows aberrant migration of glycosylated forms as reported earlier (Capellari et al. 2000) , whereas PrP 2~ shows a preponderance of the diglycosylated form with a barely detectable signal for unglycosylated PrP and a significantly reduced signal for the complex glycosylated form (Fig. la, lanes 1, 4, and 7) .
To evaluate if the reduction in glycosylated forms of PrP 2~ is due to increased degradation or block of transport at a site proximal to the Golgi, lysates of PrP c, PrP 2~176 and PrpZ~ were digested with endoglycosidase-H (endo-H) and the N-glycanase PNGase-F. The enzyme endo-H cleaves core glycans that have not been processed by Golgi enzymes, and indicates that the protein has not been transported past the cis-Golgi. The enzyme PNGase-F, on the other hand, cleaves all N-linked glycans. Following treatment of cell lysates with these enzymes, the glycosylated forms of PrP c are not affected by endo-H, indicating maturation and acquisition of complex glycans in the Golgi region (Fig. la,  lane 2) . A similar observation is noted for PrP 2~176 (Fig. la,  lane 5) , indicating that even though the glycans of this mutant PrP form are abnormal, these have been modified by Golgi enzymes and have therefore exited the ER. In contrast, PrP z~ shows partial but significant sensitivity to endo-H treatment, indicating that this protein is not transported beyond the ER/ct~-Golgi region (Fig. la, lane 8, Fig. lb) . Treatment with PNGase-F cleaves glycans of all three PrP forms, and the unglycosylated polypeptide migrates at 27 kDa as expected (Fig. la, lanes 3, 6, and 9 ). Biogenesis of P r P 2~ at early time points following synthesis was evaluated by labeling PrP c and PrPZ~ with trans-[35S]-methionine for 30 min followed by chase in normal medium for 2, 4, and 6 h, respectively (Fig. 2) . At the end of each time point, cells were washed with PBS, lysed in non-ionic detergents, and lysates were subjected to immunoprecipitation with anti-PrP antibodies 8H4 and 3F4 (Fig. 2, upper and lower panels, respectively) . Following the pulse, unglycosylated and two immature glycoforms of PrP c are detected. Only a small amount of PrP c acquires complex glycans, indicating that most of the newly synthesized PrP c is still within the ER after 30 rain of synthesis (Fig. 2, lane 1) . Following the chase, as expected, PrP c glycoforms exit the ER and are transported along the secretory path, acquiring complex glycans in the Golgi on their way to the plasma membrane (Fig. 2, lanes 1-4) . PrP 2~ on the other hand, does not acquire mature glycans and appears to undergo degradation within 2 h of chase (Fig. 2, lanes 5-8) .
Overnight labeling of PrP c, PrP 2~176 and prp2~ with tran-[35S]-methionine followed by immunoprecipitation with 3F4 and 8H4 as above shows the expected glycoforms of PrP c (Fig. 3, lanes 1 and 4) , and abnormally glycosylated forms of PrP 2~176 as reported previously metabolically labeled with Tran35S-methionine for 30 min were chased for 2, 4, and 6 h, and cell lysates were subjected to immunoprecipitation with 8H4 and 3F4. Radiolabeled PrP was visualized following SDS-PAGE-fluorography. Immediately following the pulse, the unglycosylated and core glycosylated forms of PrP c are detected with both antibodies (lane 1). PrP 2~ lysates show a similar glycoform profile, though the unglycosylated form is under-represented (lane 5). Following the chase, PrP c forms mature and acquire complex glycans, whereas PrP 2~ degrades rapidly with little or no signal detected after 2 h of chase (lanes 1-4 and ,5-8, respectively) . Control cells do not show detectable PrP when immunoprecipitated with 8H4 (vector lane) (Fig. 3 , lanes 2 and 5; Capellari et al. 2000) . In contrast, PrP 2~ shows a prominent diglycosylated form and less prominent mono-and complex glycosylated forms when immunoprecipitated with 3F4 (Fig. 3, lane 3) , and minimal reactivity with 8H4 (Fig. 3, lane 6) .
Together, the above results show that in contrast to PrP c and PrP 2~176 PrP 2~ is retained in the ER of transfected cells. Although a small amount escapes the ER and acquires endo-H resistant glycans (Fig. la, lane 8 and Fig. lb) , majority of the protein remains in the ER following synthesis. Non-reactivity of P r P 202N with anti-PrP antibody 8H4 suggests that the epitope for this antibody is unavailable due to misfolding of the protein as reported for another mutant PrP form (Gu and Singh 2004) .
Mutant P r P 202N Forms Detergent-Insoluble Aggregates in Cells
To determine if PrP 202N forms detergent insoluble aggregates as noted for certain other mutant PrP forms (Singh et al. 2005) , PrP c and prp2~
were lysed in non-ionic detergents and subjected to differential centrifugation. Under these conditions, normal PrP c partitions in the detergent soluble low speed (S1) and high speed supernatant fractions ($2), whereas detergent insoluble PrP is detected in the high speed pellet fraction (P2). Thus, cell lysates were centrifuged at low speed to pellet nuclei and cell debris (P1), and clarified supernatant fractions were divided into two parts. One was kept aside (S1), whereas the other was ultracentrifuged at 100,000 g for 1 h to isolate the high speed detergent soluble supematant ($2) and detergent insoluble pellet (P2) fractions. Proteins from all cells with anti-PrP antibodies 3F4 and 8H4. Immunoreaction of Triton permeabilized prpC-cells with 3F4 and 8H4 shows typical reaction o f PrP c on the plasma membrane and in the Golgi area (Fig. 6a, panels 1 and 2) . In Prp2~ on the other hand, 'curly' deposits of PrP that imrnunoreact with 3F4 and 8H4 are detected in the -3) , whereas a significant amount of PrP 2~ partitions in the detergent insoluble P2 fraction (lanes [4] [5] [6] fractions were precipitated with cold methanol and resolved by SDS-PAGE followed by immunoblotting with anti-PrP antibody 3F4. As expected, most of PrP c is detected in the detergent-soluble fractions S1 and $2, with minimal amounts in the P2 fraction (Fig. 4, lanes 1-3) . In contrast, most of PrP 2~ fractionates in the detergent-insoluble P2 fraction (Fig. 4, lanes 445) . Thus, PrP 2~ retained in the ER assumes an aggregated form, much like disease-associated PrP so. However, unlike PrP sc, PrP 2~ is not resistant to digestion by proteinase K (data not shown). Intracellular accumulation of PrP 2~ results in upregulation of the ER chaperone calnexin ( were exposed to 100 IxM of H2Oz for 30 min, fixed, and immunoreacted with 3F4-anti-mouse-FITC. The nuclei were visualized by staining with Hoescht. As opposed to prpC-cells, a significant number of prp2~ cells show apoptotic nuclei when exposed to H202 (arrows). Bar: 10 ~tm perinuclear region, suggestive of ER localization (Fig. 6a , panels 3, 4, and 4 inset). To confirm ER localization of PrP 2~
Triton permeabilized PrP 2~176 and prp2~ were co-immunostained for PrP and the ER specific protein calnexin (Fig. 6b) . While PrP 2~176 cells do not show colocalization of PrP and calnexin signals (Fig. 6b, panels 1-3) , PrpZ~ show co-immunostaining of PrP and calnexin when immunoreacted with either 3F4 or 8H4 followed by anti-calnexin (Fig. 6b , panels 4-6 and 7-9, respectively). As noted in Fig. 6a , PrP immunoreactive material shows a curly appearance with both 3F4 and 8H4 antibodies (Fig. 6b,  panels 4 and 7) .
Together, the above results show that PrP 2~ aggregates in the ER and assumes a 'curly' or a fibrillar appearance.
Cells Expressing PrP 2~ are Sensitive to Oxidative Stress
To evaluate if intracellular PrP 2~ aggregates increase the susceptibility of cells to oxidative stress, PrP c and PrP 2~ cells were exposed to 100 p.M H202 for 30 min, fixed, permeabilized, and immunoreacted with 3F4-anti-mouse-FITC followed by Hoescht. In contrast to prpC-cells that show minimal cell death following this treatment, ~80% of Prp2~ cells show signs of apoptosis (Fig. 7,  arrows) . Thus, PrP 2~ expressing cells are more susceptible to oxidative damage than prpC-expressing and nontransfected M17 cells.
Together, the above data demonstrate that mutant PrP 2~ is unable to fold normally following synthesis and is retained in the ER by the cellular quality control. Accumulated PrP 202N forms 'curly' or fibrillar aggregates within the ER that are insoluble in non-ionic detergents and induce upregulation of the ER chaperone calnexin. Intracellular aggregation of PrP 2~ sensitizes the cells to oxidative stress, indicating that loss of anti-oxidant function of PrP c, gain of toxic function by aggregated PrP 2~ or a state of metabolic stress induced by intracellular PrP 2~ aggregates increases the vulnerability of cells to free radical damage. This conclusion is supported by previous reports implicating PrP c as a superoxide dismutase (Brown et al. 1997 (Brown et al. , 2002 . Accumulation of PrP z~ in the ER would compromise this function and induce ER stress, adding to the already compromised ability of the cell to deal with free radicals (Hetz et al. 2005) . Although our data is specific to PrP z~ several other mutant PrP forms share similar features of abnormal processing and loss of normal function, suggesting that compromised ability of cells expressing mutant PrP forms to cope with free radical induced damage may represent a more generalized pathway of neurotoxicity in familial prion disorders than previously believed.

{'paper_id': '21994910', 'title': 'Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms ‘curly’ intracellular aggregates', 's2_url': 'https://api.semanticscholar.org/CorpusID:21994910', 'source': 'S2ORC'}