Proteomics Analysis of Amyloid and Nonamyloid Prion Disease Phenotypes Reveals Both Common and Divergent Mechanisms of Neuropathogenesis

Prion diseases are a heterogeneous group of neurodegenerative disorders affecting various mammals including humans. Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrPC) into a pathological isoform termed PrPSc. In wild-type mice, PrPC is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrPSc typically accumulates in diffuse nonamyloid deposits with gray matter spongiosis. By contrast, when mice lacking the GPI anchor are infected with the same prion inoculum, PrPSc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate whether different host biochemical pathways were implicated in these two phenotypically distinct prion disease models, we utilized a proteomics approach. In both models, infected mice displayed evidence of a neuroinflammatory response and complement activation. Proteins involved in cell death and calcium homeostasis were also identified in both phenotypes. However, mitochondrial pathways of apoptosis were implicated only in the nonamyloid form, whereas metal binding and synaptic vesicle transport were more disrupted in the amyloid phenotype. Thus, following infection with a single prion strain, PrPC anchoring to the plasma membrane correlated not only with the type of PrPSc deposition but also with unique biochemical pathways associated with pathogenesis.


■ INTRODUCTION
Prion diseases, also known as transmissible spongiform encephalopathies or TSEs, are a group of rare neurodegenerative disorders that affect humans and other mammals. 1 Animal forms of prion disease include sheep scrapie, chronic wasting disease in deer and bovine spongiform encephalopathy in cattle. 1 Human prion disorders include familial, sporadic and variant Creutzfeldt−Jakob disease (CJD), as well as kuru and Gerstmann− Straüssler−Scheinker (GSS) syndrome. A critical event in prion pathogenesis is the conversion of host-encoded prion protein (PrP C ) into a misfolded isoform, termed PrP Sc , which is widely believed to be the primary component of the infectious prion agent. Pathogenic PrP Sc has the same amino acid sequence as PrP C but has significantly more β-sheet structure and is no longer monomeric. 2,3 Variable disease phenotypes induced by different prion strains 4−6 are often attributed to specific PrP Sc conformations, 7 perhaps defined by specific interactions between amino acid side-chains within individual β strands. 8 Although no direct link between a specific structural motif in PrP Sc and a specific in vivo prion phenotype has been made, PrP C anchoring to the plasma membrane has been shown to profoundly influence prion disease phenotypes. 9−11 PrP C is normally attached to the plasma membrane by a glycosylphos-phatidylinositol (GPI) anchor. 12 When wild-type C57BL/10 mice are infected intracranially with the RML mouse prion strain, PrP Sc accumulates in diffuse nonamyloid deposits and brain pathology is distinguished by the hallmark gray matter spongiosis for which TSEs are named. Conversely, when transgenic mice homozygous for PrP C lacking the GPI anchor (Tg44 or "anchorless" mice) are infected with the same RML strain, the incubation time is longer and PrP Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. 9−11 Similar amyloid PrP Sc deposition and disease resulting in death has been observed in humans with an aberrant stop codon in the C-terminal portion of the PrP sequence, resulting in the generation of PrP lacking the GPI anchoring group as well as some amino acid residues. 13−16 Thus, GPIanchoring of PrP C to the plasma membrane appears to be a primary factor determining the type of PrP Sc deposition and the development of brain pathology.
Despite the neuropathological differences between these two prion disease phenotypes, the mechanisms underlying these differences remain unclear. Both anchorless and wild-type mice displayed a similar cytokine release profile when infected with the 22L prion agent, suggesting a similar inflammatory response. 17 When enriched PrP Sc samples from RML-infected wild-type or anchorless Tg44 mouse brains were compared by infrared spectroscopy, gross differences in secondary structure were minimal. 18 Furthermore, infectivity replicated to high levels in both types of mice and, when passaged back into wild-type mice, both led to prion disease typical of the original RML strain. 9 Taken together, the data suggest that the dramatic phenotypic differences between these two prion diseases is determined more by the presence or absence of the GPI anchor on PrP C rather than by any changes in strain-specific β-sheet conformation within PrP Sc .
Multiple studies have used microarray analysis to determine whether different pathways underlie differences in disease pathogenesis between mouse prion strains 19−24 or various types of human CJD. 25 A postgenomic, proteomics approach has been utilized in some studies, 26−30 several of which have focused on the identification of potential prion disease biomarkers (refs 26, 31, and see ref 32 for review). However, there are no genomic or proteomic studies directly comparing two different prion diseases induced by the same prion strain. In the absence of any strain-dependent phenotypic changes, such an analysis could provide useful insights into the general pathways which underlie different prion disease phenotypes.
In the present study, wild-type C57BL/10 and anchorless Tg44 mice were infected intracranially with the RML mouse prion strain. Brain homogenates from clinically positive mice were profiled for gross differences between the nonamyloid (C57BL/10 mice) and amyloid (Tg44 mice) disease phenotypes by utilizing mass spectrometry to identify changes in protein abundance. Proteomic comparison of these two disease phenotypes allowed us to distinguish the biochemical aspects of prion pathogenesis that were likely related to a generalized host inflammatory response versus those that may be more specific to each disease model.

Histopathology and Immunohistochemistry
The protocols used were described previously. 9 Briefly, PrP staining was done using an avidin−biotin complex immunoperoxidase protocol using anti-PrP antibody D13 (In-Pro Biotechnology, South San Francisco, CA, U. S. A.). Staining with a 1% Thioflavin S solution was done as previously described. 11

Preparation of Mouse Brain Homogenates
The mice used in this study were housed at the Rocky Mountain Laboratories (RML) in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and the research protocols used were approved by the NIH RML Animal Care and Use Committee. Uninfected wild-type mice (C57BL/10SnJ) were obtained from Jackson Laboratories (Bar Harbor, ME, U. S. A.). Transgenic mice homozygous for PrP C without the GPI anchor (Tg44) were described previously. 9 Six to eight week old mice were inoculated intracerebrally as described 9 with 50 μL of a 1% brain homogenate of RML containing 0.7−1.0 × 10 6 ID 50 . Animals were then observed daily for onset and progression of TSE disease. Mice were euthanized at the time of advanced clinical disease as described previously 33 and this was defined as the incubation period. Wild-type C57BL/ 10 mice developed clinical signs with a mean incubation time of 148 ± 9 days postinfection (dpi), whereas Tg44 mice developed clinical disease with a mean incubation time of 318 ± 16 dpi. For age-matched controls, uninoculated 188 day old C57BL/10 mice and 401 day old Tg44 mice were used. For all mice, brain samples were collected and flash frozen in liquid nitrogen then kept at −80°C until processed. Brain tissue was homogenized as a 20% (w/v) brain homogenate in 0.01 M Tris, pH 7.2, using a mini bead beater (Biospec, Inc.), then vortexed for 1 min followed by 1 min sonication in a Misonix cuphorn sonicator and kept at −80°C until needed.

Western Blotting
Brain homogenates analyzed without proteinase K (PK) treatment were diluted from 20% to 2.5% and boiled in Trisbuffered saline, pH 8, and sample buffer (Life Technologies) with 0.5% β-mercaptoethanol. Brain homogenates treated with PK were diluted to 6% in the presence of 0.1 M Tris, pH 8.3, 1% Triton X-100, 1% sodium deoxycholate, then digested with 125 μg/mL PK for 1 h at 37°C and then boiled in an equal volume of sample buffer. Portions of each sample (4 μL) were separated by SDS-PAGE on a 10% Bis−Tris gel with MES running buffer (Life Technologies) and then transferred to an Immobilon PVDF-FL membrane (Millipore) by wet transfer using Towbin's buffer. Samples were probed for PrP with mouse monoclonal antibody 6D11 (Covance Inc.) at a dilution of 1:15 000 in TBST buffer for 1 h. 6D11 recognizes residues 93−109 of mouse PrP. Antivimentin antibody (Origene #TA307358) was used at 1:2000. Anti-apoE antibody (Calbiochem #178479) was used at 1:1000 and antiactin (abcam #ab1801) was used at 1:1000. Secondary antibodies (Li-Cor Biosciences) were IRDye 800CW or IRDye 680CW anti-mouse, anti-rabbit or anti-goat, used at a 1:15 000 dilution in TBST buffer for 40 min. Imaging was performed with an Odyssey imaging system (Li-Cor) using the 800 and 680 channels.

In-Gel Trypsin Digestion
The in-gel tryptic digest procedure was conducted using 16 individual brains with 4 from each biological group: (1) RMLinfected Tg44 mice, (2) age-matched uninoculated Tg44 mice,  35 with the constraints that only MASCOT ion scores of ≥30 and only peptides of ≥7 amino acids in length were considered in these initial calculations. The resulting 2026 protein identifications were further filtered to require at least 2 unique peptides and a protein group probability of ≥0.9 36 for a positive identification. Additionally, each peptide was limited to only one protein identification such that the number of protein groups equaled the number of protein identifications. The list of 1567 proteins identified according to these criteria is provided as Supporting Information Table 1.

Spectral Counting
Two data sets were derived from the original 1567 protein identifications, one from the C57BL/10 mice and the other from the Tg44 mice. For label-free quantitation, 37,38 the 1567 protein identifications (Supporting Information Table 1) were normalized by dividing the spectral count (SpC) for each protein from each mouse by the mean of all proteins from that individual mouse. Once normalized, a sum was calculated for each protein for each group of mice (n = 4), and the fold change for each protein was calculated by dividing the sum for the four scrapieinfected mice by the sum from the four uninfected mice. All calculations were done using Microsoft Excel 2010. A set of ρ values was obtained by running an analysis of variance (ANOVA; Partek Genomics Suite v6.6) for the comparisons of interest, and multiple test corrected using the false discovery rate (FDR) method of Benjamini and Hochberg. 39 Any proteins with a SpC average below 5 in both the infected and uninfected groups were flagged as below detection because it has been reported that mean counts of less than 5 spectra are often unreliable for quantitation. 37,38 In order to identify the proteins that were most affected by prion infection in each mouse line, an ascending rank order analysis was used. Individual rankings were derived based upon the combination of greatest fold change, lowest ρ value, lowest coefficient of variation, and spectral counts from the two conditions compared (i.e., uninfected versus infected brain homogenate). All four criteria were thus given equal weight in the analysis and were used to estimate changes in relative protein abundance between RML-infected brain homogenates and their respective uninfected controls for each mouse strain. The resulting four ranks for any given protein were summed and divided by the square of their sum to produce one number from all ranks. This final number was then ranked in descending order to produce the overall reported rank. Thus, the protein with the best result across all criteria produced a rank of one, followed by the rank of two for second best and continuing for each of the 1567 proteins identified. Based on the final protein rankings, the top 100 proteins were used for subsequent IPA analysis (Supporting Information Table 2).

Ingenuity Pathway Analysis
Functional analyses and pathways were examined with Ingenuity Pathway Analysis (IPA,Ingenuity Systems, www.ingenuity.com). The top 100 ranked proteins from the Tg44 and the C57BL/10 lists, most of which had ρ values of ≤0.05, were evaluated against the Ingenuity Knowledge Base. The IPA database organizes lists of genes into graphical representations based upon text-mining of published interactions between the genes of interest and various predefined networks, disease classifications, canonical pathways or biofunctions. The scores assigned to the data sets by IPA, based upon their degree of overlap with the experimental data, are the negative exponent of the right-tailed Fisher's exact test result. The ρ values reported estimate the probability that each disease classification or pathway assigned to the data could have been determined by random chance. For example, a ρ value of 0.01 would indicate a 1% false discovery rate for a set of protein associations with a particular classification. A random set of 100 proteins from the Swiss-Prot murine database generated IPA ρ values of 7.1 × 10 −5 −1.5 × 10 −2 (data not shown). Therefore, we considered the association of proteins with an IPA ρ value of ≤5 × 10 −5 with a particular molecular functional group to be significant and outside of the realm of random chance.

Inoculation of C57BL/10 and Tg44 Mice with the RML Mouse Scrapie Strain
After intracranial inoculation of RML mouse scrapie, significant gray matter spongiosis was observed only in RML infected C57BL/10 mice ( Figure 1A inset). C57BL/10 mice expressing GPI-anchored PrP C also exhibited diffuse PrP Sc deposits in the brain ( Figure 1A) and had a disease incubation time of approximately 150 days. By contrast, the same inoculum into Tg44 mice, which express anchorless PrP C , led to the deposition of PrP Sc in large plaques ( Figure 1B) with little or no gray matter spongiosis ( Figure 1B inset) and an incubation time of approximately 320 days. When brain sections were stained with the amyloid-binding dye thioflavin S, the diffuse PrP Sc deposits in C57BL/10 mice were negative (data not shown), whereas the large plaque-like PrP Sc deposits in Tg44 mice were positive ( Figure 1C). The disease phenotype of RML infected C57BL/10 mice will therefore be referred to as nonamyloid, whereas that of RML infected Tg44 mice will be referred to as amyloid. Consistent with previous results, 9,10 these data show that the presence or absence of a GPI anchor on PrP C can induce two markedly distinct prion diseases. Moreover, the neuropathological differences observed between the two mouse models suggest that the biological pathways underlying prion pathogenesis in these mice fundamentally differ.

Identification and Quantitation of Proteins Expressed in the Brains of Mice with the Amyloid and Nonamyloid Disease Phenotypes
In order to gain more insight into these potential differences, we adopted a proteomics approach. Brain homogenates from infected versus uninfected Tg44 and C57BL/10 mice were examined, each with four biological replicates, for a total of 16 individual brains. A representative Coomassie blue stained gel loaded with approximately 50 μg of protein for each replicate demonstrated a similar protein staining profile for each sample ( Figure 2A). As outlined in Figure 2B, the initial list of proteins identified was set to a false discovery rate (FDR) threshold of ≤1%, 35 resulting in 2026 nonredundant protein identifications. These data were additionally filtered so that only proteins identified by ≥2 unique peptides and a protein group probability of ≥0.9 based upon the Protein Prophet algorithm 36 were selected for further analysis. The resulting protein list contained 1567 high-confidence protein identifications (Supporting  Information Table 1), derived from a total of 12 834 unique peptides across all of the biological groups. The number of protein identifications was similar between groups ( Figure 2C, left panel) with the vast majority of proteins common to all four groups ( Figure 2C, right panel), indicating good consistency in protein sampling.

Relative Abundance of PrP Varies between Biological Groups
The relative apparent abundance of the proteins between sample groups was examined with the goal of comparing the global effects of prion infection in the amyloid and nonamyloid disease phenotypes at the end-stage of clinical disease. In order to semiquantitatively compare the relative abundance of proteins between the groups, spectral counting was utilized. Spectral counting is often used as a practical, effective approach for estimating the relative abundance of proteins between biological samples 37,38,40 and is the preferred method for quantifying profiling data from ion trap mass spectrometers such as the one used in this study. 41 Figure 1. Infection of C57BL/10 and Tg44 mice with the RML prion strain yields two distinct disease phenotypes. (A) Immunostaining of RML infected C57BL/10 brain tissue with D13 prion antibody shows diffuse PrP Sc deposition in most regions of the brain in addition to spongiform degeneration. (B) Immunostaining of RML infected Tg44 brain tissue with D13 prion antibody shows PrP Sc deposition in the absence of spongiform degeneration. (C) Staining of the brain hippocampal region with the amyloid-specific fluorescent dye Thioflavin S confirms the presence of amyloid plaque in RML infected Tg44 brain. Thioflavin S staining is negative in uninfected Tg44 mice (inset).
As expected, PrP was identified as a protein with an altered relative abundance when compared to uninfected controls. Based upon spectral counts, there was an approximate 5-fold increase in PrP C in uninfected C57BL/10 mice when compared to uninfected Tg44 mice ( Figure 3A). Our semiquantitative analysis using spectral counts was therefore consistent with previous findings suggesting that uninfected Tg44 mice express approximately 8-fold less PrP C relative to uninfected C57BL/ 10 mice. 9 The amount of PrP in brain homogenates from the two prion-infected mouse lines was also significantly different. In the absence of PK treatment, total PrP levels (i.e., PrP C and PrP Sc ) were significantly higher in mice with the amyloid disease phenotype than in any other sample group, demonstrating an apparent 91-fold higher concentration of total PrP (Table 1) when compared to age-matched controls ( Figure 3A). By contrast, a 2-fold increase in total PrP was observed with the nonamyloid disease phenotype (Table 1) when compared to their uninfected controls ( Figure 3A). These differences in the total amount of PrP were confirmed by immunoblot analysis (Supporting Information Figure 1A). After PK digestion, protease-resistant PrP Sc was found only in the infected samples ( Figure 3B). The increased levels of total PrP in both strains of infected mice therefore appears to represent the generation and accumulation of PrP Sc .

Neuroinflammation and Complement Activation Are Common to Both Prion Disease Phenotypes
Each of the 1567 identified proteins was ranked for both the amyloid and nonamyloid phenotypes and the top 100 ranked proteins (Supporting Information Table 2) from each data set were compared. These proteins had the strongest association with each disease phenotype and were thus the primary focus of our subsequent data analysis. Only 17 of the top 100 proteins  were common to both disease phenotypes. In every instance, proteins that showed a significant fold increase or decrease in the amyloid disease phenotype mice showed similar fold increases or decreases in the nonamyloid disease phenotype (Table 1). Consistent with the gliosis observed in both prion disease models, 9,10 the astrocyte protein glial fibrillary acidic protein  (GFAP) was increased 5.8 and 8.9 fold in the amyloid and nonamyloid disease phenotypes, respectively (Table 1). 42 Proteins such as vimentin, α-2-macroglobulin, and α-2-HSglycoprotein, all of which have been linked to neuroinflammation in prion as well as other neurodegenerative diseases, 43−48 were increased anywhere from 2.8−26.7 fold in both disease phenotypes ( Table 1). The increased levels of vimentin in prion infected mice was further confirmed by immunoblot analysis (Supporting Information Figure 1B). Finally, multiple complement factors which have been linked to neuroinflammatory responses 49 also had elevated spectral counts in both prion disease models with increases in relative abundance ranging from 7.8−27.6 fold (Table 1). It was unlikely that the increased relative abundance of these proteins was due to age-related differences between C57BL/10 and Tg44 mice because they were present at comparable levels in uninfected controls from both strains of mice ( Figure 4A−F). Thus, the similar expression levels of these proteins in mice with both the amyloid and nonamyloid disease phenotypes may reflect a similar response and activation of astroglia and microglia to the two different types of brain damage that result from RML prion infection in Tg44 and C57BL/10 mice.

Increased Levels of Proteins Associated with Fatty Acid Metabolism in Both Prion Disease Mouse Models
In mice with the amyloid disease phenotype, expression levels of several key apolipoproteins (apoD, apoE, apoJ) as well as two other proteins involved in lipid transport, peroxisomal multifunctional enzyme type 2 and low density lipoprotein receptor related protein 1 (LRP1), were elevated when compared to uninfected age-matched controls ( Table 2). These proteins were generally much higher ranked in the amyloid-associated data set than they were in the nonamyloid data set. The greater abundance of these proteins in mice with the amyloid phenotype was further confirmed by immunoblot for one of them, apoE (Supporting Information Figure 1C). Furthermore, the differences in relative abundance did not appear to be due to agerelated differences between Tg44 and C57BL/10 mice because these proteins were present at comparable levels in uninfected controls from both strains of mice ( Figure 4G−I). Thus, these data suggest that, although lipid metabolism and transport are altered in both the amyloid and nonamyloid phenotypes, this disruption is more strongly linked to the amyloid form of disease.

Molecular and Cellular Functions Disrupted in Prion-Infected Mice
In order to further differentiate the two prion disease phenotypes, the top 100 ranked proteins in each data set were analyzed using Ingenuity Pathway Analysis (IPA) which maps the molecular and cellular pathways associated with those proteins. Our initial analysis showed that individual proteins were often associated with multiple functional processes, making it difficult to determine which proteins and pathways were most specifically relevant to prion disease. Therefore, to identify the potential molecular mechanisms involved in pathogenesis, we focused our initial analysis on the molecular and cellular function categories, particularly on those subcategories which contained the highest percentage of unique proteins (Table 3). Using these criteria, cell death and survival was the top scoring group in the nonamyloid phenotype, with a higher percentage of unique proteins when compared to the other groups (29% versus 3−8%, see Table 3). By contrast, in the amyloid phenotype, the highest percentage of unique proteins was associated with the molecular transport category (23%) followed by cell death and survival at 14% (Table 3). Thus, whereas cellular pathways related to cell death and survival were associated with both the amyloid and nonamyloid disease phenotypes, pathways involved in molecular transport were most strongly implicated in the amyloid disease phenotype.  In mice with the amyloid disease phenotype, the 39 proteins implicated in molecular transport (Table 4) were involved in the binding and transport of multiple molecules including amino acids, metals, and ADP/ATP. Interestingly, many of these proteins were associated with metal-dependent processes, many of which involved calcium ( Figure 5A). Further analysis revealed another protein known to bind calcium, calcineurin subunit type B1, that had not been identified by the IPA analysis. Finally, multiple proteins involved in calcium transport, regulation, and homeostasis were also linked to molecular transport in the amyloid phenotype (Supporting Information Table 3). Thus, our data suggest that metal ion transport, and in particular calcium homeostasis, is disrupted in the amyloid form of prion infection. Interestingly, many of the proteins in the molecular transport category were also involved in synaptic vesicle transport, a process known to be regulated by calcium. 50 They included synaptosomal-associated protein 25 (SNAP25) and syntaxin 1A, which are two of the three proteins present in a complex necessary for synaptic fusion and neurotransmitter release, as well as several proteins involved in synaptic vesicle exocytosis 50,51 including syntaxin 1B, rabphilin 3a, and members of the Ras-related proteins ( Table 4). All of these synaptic vesicle proteins were present in lower abundance when compared to Low-density lipoprotein receptor-related protein 1 ↑ 2. noninfected controls, suggesting significant disruption to synaptic vesicle transport in prion-infected mice with the amyloid disease phenotype.

Changes in Cell Death and Survival Pathways Suggest Potentially Different Mechanisms of Cell Death in the Nonamyloid versus Amyloid Phenotypes
The highest scoring molecular function category associated with the top 100 proteins in mice with the nonamyloid disease phenotype was cell death and survival, with 48% of the 100 input proteins linked to this category (Table 5). Of those, 29% (14 out of 48) were uniquely associated with cell death and survival and were not found in any of the other categories ( Table 3). The cell death and survival category proteins had diverse functions including protein folding, cytoskeletal organization, RNA processing, apoptosis, and intracellular calcium regulation. However, there was an increase in the relative levels of three proteins associated with the mitochondrial inner membrane. Two of these, the mitochondrial inner membrane protein (mitofilin) and heat shock protein HSPA9, are part of the mitochondrial inner membrane. The third is apoptosis-inducing factor 1 (AIF1), which is anchored to the mitochondrial inner membrane. 52 Finally, the fold expression of several proteins associated with the ubiquitin/proteasome pathway was also increased. These included heat shock protein HSPA5 (or BiP), which interacts with proteins involved in endoplasmic reticulum associated degeneration (ERAD), as well as the protein chaperonin TCP1 (Table 5). Thus, the data are consistent with disruptions to mitochondria, ER and the ubiquitin/ proteasome pathway as part of the process of cell death in the nonamyloid prion disease phenotype. Cell death and survival was also the second highest scoring IPA functional category in prion-infected mice with the amyloid phenotype and contained 42 of the top 100 input proteins, 14% of which uniquely segregated into this category ( Table 6). Many of these proteins, including annexin A2, gelsolin, syntaxin 1A and SNAP25, overlapped with those proteins associated with calcium binding and molecular transport ( Figure 5B). This overlap suggested a strong link between perturbations in calcium homeostasis and cell death in the disease with the amyloid phenotype. Interestingly, however, none of the mitochondrial or ubiquitin/proteasome pathway proteins identified in the mice with the nonamyloid phenotype were found in the top 100 ranked proteins in mice with the amyloid disease phenotype. In fact, there was minimal overlap with the proteins associated with cell death and survival in the nonamyloid versus the amyloid disease phenotypes (9 out of 90, or 10%). Moreover, several of these 9 proteins, including GFAP, vimentin, complement, and α-2-macroglobulin have previously been associated with a strong inflammatory response in prion diseases 27,49,53 and are considered global neuroinflammatory markers, indicating that they are not functionally restricted to pathways mediating cell death. Thus, our data suggest that different cell death pathways may predominate in the amyloid and nonamyloid disease phenotypes.

■ DISCUSSION
Previous genomic studies in prion-infected mice have suggested that disruptions to metal binding, calcium homeostasis, synaptic transmission, lipid metabolism, and protein folding/degradation are involved in the pathogenesis of prion diseases. [19][20][21][22][23]53 Similarly, genomic and proteomic analysis of tissue from human CJD patients has suggested that disruptions to molecular transport, calcium homeostasis, mitochondria, synapses and glucose metabolism may be involved in the pathogenesis of CJD. 25,26,29,54 Our proteomics analysis of two different models of mouse prion disease also implicated imbalances in metal binding, calcium homeostasis, mitochondria, synaptic transmission, lipid metabolism, and molecular transport as factors in the late stages of prion disease. However, unlike previous studies, our comparison of the two distinctive prion disease phenotypes induced by a single prion strain has also allowed us to distinguish which pathways were more strongly linked to amyloid or nonamyloid forms of prion disease as well as which likely represented general responses to prion infection.
Our analysis primarily focused on the 100 proteins that were linked most strongly to either the amyloid or nonamyloid disease phenotype. Of these, only 17 were common to both disease phenotypes and most had some link to neuroinflammation (Table 1). This is consistent with previous studies showing that multiple genes and proteins associated with the inflammatory response are upregulated in different types of mouse 22,23,27,55 and human 25,26 prion disease. Astrocytes which have been activated in response to damage or infection produce complement factors and increase the expression levels of both vimentin and GFAP, 45,56 all of which are present at increased levels in our two mouse models ( Table 1). The increased abundance of α-2macroglobulin may be related to its ability to act as a carrier protein for interleukin-6 46 or to remove proteinases at the site of inflammation. 57 Likewise, α-2-HS-glycoprotein, an acute phase protein which binds calcium and has anti-inflammatory properties, was also increased in abundance in both disease models. 48 Although the magnitude of the changes in fold expression was not always identical between the two phenotypes, proteins which were increased or decreased in mice with the amyloid phenotype mice were also increased or decreased in mice with the nonamyloid phenotype (Table 1).
Interestingly, many of these same proteins are also increased or decreased in abundance in other human neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease. For example, upregulation of vimentin has recently been described in neuronal cells of AD patients as part of a damage response mechanism, whereby the neurons attempt to repair synapses and re-establish dendritic branches lost during the disease process. 43 α-2-macroglobulin has been found in senile AD plaques, 47 whereas α-2-HS-glycoprotein has been found, albeit at decreased levels, in the cerebrospinal fluid of Alzheimer's patients. Complement factors (Table 1) have also been proposed Figure 5. The majority of molecular transport proteins disrupted in prion-infected Tg44 mice were associated with metal-dependent processes. (A) Relative distribution of the metal-associated molecular transport proteins from Table 4. (B) Venn diagram illustrating the overlap between molecular transport (Table 4) and cell death and survival (Table 6) in the amyloid phenotype and their overlap with known calcium binding proteins from each category.
to play a role in both Parkinson's and Alzheimer's diseases 32 When taken together, the data suggest that the changes in the relative abundance of these proteins during disease may represent similar, general responses related to the damage in the brain which occurs during neurodegeneration rather than a direct response to the deposition of PrP Sc as amyloid versus nonamyloid. LMNB1 MAPT An increased relative abundance of several lipoproteins was also observed in both disease phenotypes (Table 2). Notably, the lipoproteins apoD, apoE and clusterin (apoJ) as well as the low density lipoprotein receptor-related protein (LRP1), were elevated in both data sets but present at higher levels in the amyloid disease phenotype (Table 2). ApoE consistently copurifies with PrP Sc isolated from infected animal tissue 34,58−60 and elevated levels of clusterin have been observed in the hippocampus of prion infected mice, 27,28 raising the possibility that these molecules have a functional role in prion pathogenesis.
The ability of clusterin to inhibit the formation of amyloid 61 as well as the link between LRP1 and the apoE and α-2macroglobulin-mediated clearance of β-amyloid 62,63 may explain their elevated levels in the amyloid disease phenotype. It is possible that other clusterin functions, such as its ability to prevent apoptosis induced by the mitochondrial release of cytochrome C, 64 may also be relevant in the nonamyloid disease phenotype where proteins necessary for the integrity of the mitochondrial crista (Table 5) are also elevated. Overall, our data suggest that disruption of cholesterol and lipid metabolism and Growth factor receptor-bound protein 2 ↓ −2. alterations in the mechanisms for the clearance of misfolded proteins occur in both amyloid and nonamyloid forms of prion disease. However, these changes are more pronounced in the mice with the amyloid disease phenotype. Thus, the presence of amyloid may exacerbate disruptions in lipid metabolism and protein clearance. Consistent with this idea, apoD and apoE have been reported as candidate biomarkers for AD and prion disease 32 and are known to colocalize with amyloid plaque in both Alzheimer's disease 65,66 and cerebral amyloid angiopathy. 67 Moreover, the ability of clusterin to inhibit the formation of amyloid 61 as well as the link between LRP1 and the apoE and α-2-macroglobulin-mediated clearance of Alzheimer's disease βamyloid 62,63 may explain their elevated levels in the disease phenotypes where amyloid is a prominent feature.
In addition to the perturbation of the lipid transport proteins that we observed in both phenotypes, other molecular transport proteins were also found to have significantly changed in abundance in mice with the amyloid disease phenotype (Table  4). Approximately one-third of these are known to bind metals such as iron, zinc, manganese, copper and magnesium ( Figure  5A) indicating a metal imbalance that could contribute to the buildup of reactive oxygen species and reduction of superoxide dismutase activity that has been observed during prion infection. 68 Interestingly, multiple proteins were involved with calcium transport, regulation, and binding (Supporting Information Table 3), suggesting a significant disruption to calcium homeostasis in mice with the amyloid disease phenotype. Divalent calcium ions are universal signaling molecules with cells maintaining an approximately 20 000-fold lower concentration of divalent calcium ions in the intracellular compartment compared to the extracellular compartment. 69 Imbalances in this tightly controlled gradient can trigger a wide variety of cellular processes 69 and dysregulation of calcium has been implicated in the pathogenesis of prion disease. 70 Our data suggest that in addition to dysregulation of calcium homeostasis, synaptic vesicle transport may also be impaired in the mice with the amyloid phenotype. Synaptic vesicle transport and exocytosis is triggered by an influx of calcium at the synapse leading to fusion of the soluble NSF attachment protein receptor (or SNARE) complex to the membrane and the subsequent release of neurotransmitters from the synaptic vesicle. 50 SNARE is essential for synaptic vesicle fusion to the membrane and syntaxin-1 and SNAP25, two critical proteins which comprise SNARE, are decreased in abundance in mice with the amyloid phenotype. Similarly, the levels of syntaxin 1B, Rab3a, Rab3b and rabphilin-3a, all of which participate in the fusion of synaptic vesicles, 50,51 are also decreased (Table 4). Interestingly, altered cycling of Rab3a has recently been reported in CJD 54 and alterations in synaptic vesicle fusion and recycling can lead to disruptions in neurotransmitter release and deficits in learning and memory, a phenotype which has been described in the prioninfected mice with amyloid disease. 71 Thus, our data suggest that impairment of synaptic vesicle fusion and neurotransmitter release triggered by changes in calcium homeostasis may underlie the learning and memory deficits seen in mice with the amyloid disease phenotype. SNARE proteins have also been associated with neurodegeneration in CJD, 25,72 and synaptic degeneration precedes neurodegeneration in prion infected mice. 73 Thus, SNARE proteins may also be involved in neurodegenerative processes. Consistent with this hypothesis, IPA analysis linked syntaxin-1 and SNAP25 to cell death and survival pathways only in mice with the amyloid disease phenotype (Table 6). However, synaptic degeneration has been observed in multiple mouse prion disease models, 73,74 including the RML strain used for the current studies. 75 Furthermore, a decrease in SNAP25 and syntaxin has been reported in RML-infected wild-type mice. 20,75,76 Thus, contrary to the present data, synaptic transport can also be impaired in nonamyloid forms of prion disease. The reason for this discrepancy is not clear but it is possible that decreased expression of SNARE proteins in the amyloid phenotype is the result of changes in synaptic vesicle fusion not linked to SNARE-mediated neurodegeneration, perhaps because of the lack of membrane anchored PrP C in these transgenic mice. This hypothesis is consistent with the lack of significant gray matter spongiosis in the amyloid disease phenotype 9−11 as well as studies showing no correlation between decreased SNAP25 and apoptosis in RML infected mice. 75 In the proteins associated with cell death and survival (Tables 5 and 6), there was very little overlap between the amyloid and nonamyloid disease phenotypes even though multiple proteins linked to calcium homeostasis were identified for both disease forms. This suggested that the dominant mechanisms underlying calcium-associated cell death may differ between the two mouse models. For example, only in the nonamyloid phenotype was there an increased abundance of both chaperonin containing TCP1 and the ER protein BiP, both of which are part of a large chaperonin complex that interacts with multiple proteins linked to ERAD (Table 5). These data are consistent with the possibility that the ubiquitin-proteasome system is involved in prion pathogenesis but that it may be more disrupted in the nonamyloid disease phenotype. 77,78 Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in prion disease. 79,80 Our data also implicated mitochondrial pathways of apoptosis in neurodegeneration but only in the nonamyloid disease phenotype. Mitochondria have been linked to apoptosis via the release of pro-apoptotic factors such as cytochrome C and AIF1 from the inner mitochondrial membrane. 52, 81,82 Disruptions in this membrane have been observed in prion infected mice in regions of synaptic degeneration. 80 Mitofilin and HSPA9, two proteins associated with the mitochondrial inner membrane organizing system (MINOS) responsible for maintaining cristae morphology, were present at higher levels only in the nonamyloid phenotype. The level of AIF1, an apoptosis inducing factor anchored to the mitochondrial inner membrane, was also elevated. In response to increased cytoplasmic calcium, mitochondrial calcium also increases leading to permeabilization of the mitochondrial outer membrane, changes in cristae morphology, and the production of reactive oxygen species. The resultant cleavage of AIF1 from the inner mitochondrial membrane allows it to translocate to the nucleus where it triggers DNA fragmentation and apoptosis, primarily in neurons. 52 The altered levels of mitofilin, HSPA9, AIF1 and multiple proteins involved in calcium homeostasis are all consistent with this mechanism of mitochondria-associated apoptosis in mice with the nonamyloid disease phenotype.
Overall, our data suggest that different mechanisms of cell death may predominate in amyloid versus nonamyloid forms of prion disease, with ERAD and mitochondria induced apoptosis implicated primarily in the nonamyloid disease phenotype when compared to the amyloid disease phenotype. This difference may be driven more by the presence of the GPI anchor on PrP C , either because of the possible function of PrP C in cell signaling and apoptosis 83 or because of the cell-surface conversion of PrP C by PrP Sc . Thus, rather than being a phenotype necessarily encoded by PrP Sc , our data suggest that the spongiform change associated with prion diseases is linked to membrane-anchored PrP C and to complex calcium-regulated changes in mitochondria and the ERAD/ubiquitin-proteasome system. The lack of gray matter spongiosis in the nonamyloid phenotype mice, despite very high levels of abnormal PrP Sc , is consistent with this hypothesis and further suggests that spongiform change is not necessarily driven by a loss of function of membrane anchored PrP C or by the amount of PrP Sc deposited. In fact, the deposition of anchorless PrP Sc as amyloid may be somewhat protective, possibly resulting in slower disease progression. This may be due to different cell death pathways being induced. Alternatively, it may simply take longer for PrP Sc amyloid to alter brain homeostasis sufficiently to trigger the mechanisms that eventually lead to neurodegeneration.
■ ASSOCIATED CONTENT * S Supporting Information Supplemental Figure 1: Western blot analysis of the changes in relative abundance of selected proteins. Supplemental Table 1: List of 1567 protein identifications with normalized spectral counts. Supplemental Table 2: List of the top 100 ranked proteins for the amyloid and nonamyloid data sets based upon the lowest p value, highest fold change, best count combination, and lowest coefficient of variation. Supplemental Table 3: Proteins involved with calcium transport, regulation, and binding in the amyloid data set. This material is available free of charge via the Internet at http://pubs.acs.org.