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 (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc 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, PrP^Sc 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, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

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 (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc 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, PrP^Sc 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, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

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 (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc 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, PrP^Sc 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, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

Varying RNA and POPG concentration does not lead to rPrP-res that can induce PrP^Sc formation in SN56 cells.

<p>(A) rPrP PMCA reactions seeded with rPrP-res and serially propagated in the presence of 30 µg/ml of RNA and 3 different concentrations of POPG lipid (POPG lanes) or in the presence of 4.4 µg/ml of POPG and 3 different concentrations of mouse liver RNA (RNA lanes) were digested with PK and assayed in duplicate by immunoblot. (B) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 30 µg/ml RNA plus 2.2 or 6.7 µg/ml of POPG lipid. (C) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 4.4 µg/ml POPG plus 15 or 45 µg/ml of RNA. (D) SN56 cells inoculated with rPrP-res (Passage 6). The concentration in µg/ml of the RNA and POPG used is indicated in the parentheses. Lanes labeled 1× indicate PMCA products generated using the original POPG and RNA concentrations of 4.4 µg/ml and 30 µg/ml, respectively. (E) SN56 cells inoculated with rPrP-res (Passage 10). Labeling is as in panel D. The first lane is a 1∶100 dilution of undigested PrP from SN56 cells. Based on a standard dilution curve of PrP^Sc derived from SN56 cells persistently infected with 22L (first 3–4 lanes in panels B-D), the limit of detection of the immunoblot for PrP is equal to ∼1–3% of the total cell equivalents loaded. For all panels, blots were developed using the anti-PrP mouse monoclonal antibody 6D11. Molecular mass markers (kDa) are indicated on the right of each panel. Irrelevant lanes have been cropped and some lanes have been rearranged for clarity, but each immunoblot panel derives from a single film exposure.</p

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 (PrP^C) into a pathological isoform termed PrP^Sc. In wild-type mice, PrP^C is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and PrP^Sc 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, PrP^Sc 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, PrP^C anchoring to the plasma membrane correlated not only with the type of PrP^Sc deposition but also with unique biochemical pathways associated with pathogenesis

Two different preparations of mouse rPrP do not spontaneously form rPrP-res capable of inducing the formation of mammalian PrP^Sc in SN56 cells.

<p>(A) The products of eleven rounds of unseeded rPrP2 PMCA were PK digested and assayed by immunoblot with the C-terminal anti-PrP rabbit polyclonal antibody R20. The emergence of a13 kDa rPrP-res2 band (closed arrowhead) can be seen in Round 11. Unsonicated rPrP2 substrate mixture in the absence (PK-) or presence (PK+) of PK is shown in lanes 1 and 2. The PK- control sample was diluted 1∶100 prior to loading. Purified mouse rPrP from residues 90–230 is shown in the last lane as a molecular mass reference. Arrow: rPrP. (B) PMCA reaction products and unsonicated rPrP2 substrate (No Sonic) were digested with eight-fold dilutions of PK and assayed by immunoblot using the C-terminal anti-PrP rabbit polyclonal antibody R20. The PK- control sample was diluted 1∶100 prior to loading (lane 1), while the digested samples were loaded undiluted. Undigested rPrP peptide 23–230 (Lane 1, arrow) and rPrP peptide 90–230 (Lane 11) are used as references for molecular mass. In both panels A and B, the non-PrP bands from ∼18–66 kDa are derived from the digestion products of BSA and PK. Open arrowhead: 16 kDa rPrP-res; Closed arrowhead: 13 kDa rPrP-res. (C-E) SN56 cells were inoculated with cell culture medium alone (Mock), brain homogenate from mice with clinical 22L scrapie (22L), 120 ng of rPrP-res, or 7 ng of rPrP-res2. Cell lysates were PK-digested and analyzed by immunoblot with the anti-PrP mouse monoclonal antibody 6D11 at Passes 0–3 (C), 0–2 (D) and passage 10 (E). All rPrP-res and rPrP-res2 samples were loaded undiluted. In panels D and E, a dilution series of a 22L scrapie-infected cell lysate was used to demonstrate the limit of detection of PrP^Sc (Lanes 1–3). For all gels, molecular mass markers (kDa) are indicated on the right. Irrelevant lanes have been cropped but each immunoblot panel derives from a single film exposure.</p

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

<div><p>During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.</p></div

C57Bl/10 mice inoculated with rPrP-res do not accumulate detectable PrP^Sc up to 626 days post inoculation.

<p>Brain and spleen samples from C57Bl/10 mice inoculated with rPrP-res, 22L mouse scrapie, vehicle alone (Neg), or a mixture of unsonicated rPrP, POPG, and RNA in inoculation buffer (No sonic) were homogenized, PK digested, and assayed by immunoblot using the anti-PrP mouse monoclonal antibody 6D11. (A) Brain and spleen, 200 dpi. (B) Brain, 365 dpi. (C) Spleen, 365 dpi. (D) Spleen, 365 dpi, secondary antibody only. Samples assayed are identical to those in panel C. The first lane shows the reactivity of the secondary antibody with purified mouse IgG heavy and light chains. (E) Brain, 626 dpi. (F) Spleen, 626 dpi. For all panels, tissue samples were loaded undiluted (UD) unless otherwise noted. A standard dilution curve of 22L mouse brain homogenate containing PrP^Sc was loaded on each gel as indicated and used to estimate the limit of detection of PrP as detailed in the Materials and Methods. A, B, and C represent individual mice assayed at each timepoint. IC = intracerebral; IP = intraperitoneal; PO = per ora. Molecular mass markers (kDa) are indicated on the right and the asterisk indicates the location of a cross-reactive proteinase K band.</p

Lack of spongiform change and PrP^Sc in C57Bl/10 mice inoculated with rPrP-res.

<p>Sagittal sections from C57Bl/10 mice inoculated with 22L scrapie, rPrP-res, or the unsonicated rPrP mixture (No sonic) stained with D13 anti-PrP antibody. A representative region of the thalamus is shown. Mice inoculated with 22L showed clear spongiform change and PrP^Sc deposition (brown stain). No spongiform change or PrP^Sc was detected in any region of the brain from mice inoculated with rPrP-res. Scale bar = 100 µm.</p

rPrP-res is detergent insoluble.

<p>Unsonicated (No sonic) or sonicated (rPrP-res) PMCA substrate mixtures were subjected to ultracentrifugation for one hour at 100,000×<i>g</i> and the resulting supernatants (SN) and pellets were assayed by immunoblot with the anti-PrP mouse monoclonal antibody 6D11 either with (+) or without (−) PK digestion. Samples were loaded undiluted (UD) or diluted 1∶10 and 1∶100 as indicated. Molecular mass markers (kDa) are shown on the right. rPrP is indicated by the arrow and the 16 kDa protease-resistant band by an open arrowhead. The band at ∼66 kDa is bovine serum albumin which was added to the samples during methanol precipitation. The asterisk indicates the location of a cross-reactive proteinase K band. Lane numbers are shown at the bottom of the gel. Total = no centrifugation.</p