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

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

Immunoblot detection of PrPres from various tissues of 22L scrapie-infected anchorless PrP tg44 mice and WT mice.

<p>Blots were developed using D13 anti-PrP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000232#ppat.1000232-Matsunaga1" target="_blank">[26]</a>. No PrPres was detected in uninoculated tg44 or WT mice. For tg44 mice (A), lane 1, mock infected tg44 mouse brain, 1.0 mg tissue equivalents. Lanes 2–6 were loaded with 0.25–0.5 mg tissue equivalents and developed using an enhanced chemiluminescence exposure of 5 seconds, and lanes 7–13 were loaded with 1.0 mg tissue equivalents and exposed for 1–2 minutes. Lane 11, liver was negative, and lane 12 was the only positive liver of nine tested. In tg44 mice, spinal cord, spleen, lymph node, intestines, kidney, and lung were also positive, and eye and skin were negative (data not shown). Note different PrPres banding pattern seen in tg44 mice: predominant band is the 18 kD unglycosylated form. Next in abundance is the 22kD monoglycosylated form. The 25kD diglycosylated form is barely visible in lanes 5, 6, 9, and 10. Dimeric PrP bands at 36kD are seen most clearly in lanes 3, 5, 6, and 9. For WT mice (B), lane 1 was loaded with 0.25 mg tissue equivalents while lanes 2–8 were loaded with 1.0 mg tissue equivalents. Blot was exposed for 2 minutes to search for possible weak bands in lanes 2–8. Lane 1 was overexposed, but showed usual PrPres banding pattern for 22L mouse scrapie brain. Results are representative of tissues from at least five mice analyzed in each group.</p

Infectivity titers in scrapie-infected WT and anchorless PrP transgenic (tg44) mice.

a<p>Following IC inoculation, tissues were analyzed at 482–654 days for tg 44 mice and 155–165 dpi for WT mice. Plasmas were analyzed from 365–596 dpi for tg44 and 155–165 dpi for WT mice.</p>b<p>Titers are expressed as log₁₀ID50/gram tissue or ml plasma <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000232#ppat.1000232-Dougherty1" target="_blank">[37]</a>. When recipient mice were inoculated with 50 µl of a 1% tissue homogenate or 50 µl of a 1/100 plasma dilution, the lower limit of detection was 2.8 log₁₀ID50/gram tissue or ml plasma. Some mice were inoculated with 50 µl of a 1/10 dilution of plasma, so the lower limit of detection was 1.8 log₁₀ID50/ml plasma. Tissues from mock-inoculated or uninoculated mice had no detectable infectivity.</p>c<p>Two tg44 mice inoculated IP were sacrificed at 441 and 692 dpi.</p>d<p>These low or negative plasma titers differed from a previous report <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000232#ppat.1000232-Trifilo1" target="_blank">[25]</a>, where titers of >5.0 and >7.0 log₁₀ID₅₀/ml were found in two anchorless PrP tg mice at 480 and 507 days post-infection with the RML scrapie strain.</p>e<p>Mice inoculated with RML scrapie strain.</p><p>Mice without footnote were inoculated with 22L scrapie strain.</p

PrP in white fat and adjacent skeletal muscle.

<p>Detection of PrPres (red staining) used D13 anti-PrP antibody. (A) Myocytes (MS) adjacent to white fat (WF) of a homozygous tg44 mouse at 375 days post-infection with 22L scrapie. PrPres staining in fat was much more intense than in muscle (arrows), suggesting a lower level of infection in muscle. Nerves (N) had minimal staining. White fat showed smaller than usual lipid lobules, probably a consequence of clinical wasting. (B) Uninfected tg44 mouse. Fat, muscle, nerve, and brown fat (BF) are negative for PrPres. Scale bars indicate 200 micrometers.</p

Effect of coexpression of 170N and 170S PrPsen on prion disease after inoculation with scrapie strains 22L or RML.

<p>a  =  expression of PrPsen relative to C57BL/10 Prnp+/+ mouse.</p><p>b  =  incubation period is average days post inoculation +/− standard deviation to disease after inoculation with 22L or RML (stock RML-81) scrapie as described in the methods.</p><p>c  =  Prnp+/− mice have only one PrP allele and this allele expresses 170N.</p><p>d  =  p<0.001 compared to Prnp+/− mice using a 1-way ANOVA with Dunnett's multiple comparison test.</p><p>e  =  After RML infection, neither Tg330 nor Tg340 were significantly different from Prnp+/− mice when compared using a 1-way ANOVA with Dunnett's multiple comparison test (P = 0.1457).</p

PMCA reactions comparing PrP-170N and PrP-170S as PrPsen substrates.

<p>Panels A, B, D, and E show immunoblots of PMCA products visualized on the LiCOR Odyssey Scanner. RML (A and B) or 22L (D and E) scrapie-infected brain homogenates diluted 1∶400 were used to seed two different brain homogenate substrates: 170N from Tga20 mice (A and D) and 170S from Tg330 mice (B and E). For all gels, lane 1 contains unseeded Tga20 brain homogenate (5 µg brain equivalent of Tga20 brain homogenate), which was used as a control to show differences in detection sensitivity (gain setting) on the Odyssey scanner. For example, gain was increased for panels A and B compared to D and E because of the lower signal obtained with the RML seed in both 170N and 170S substrates. Other lanes contain 3.5 µl of PK-digested sonicated reaction product (lanes 3–8) or unsonicated freeze–control (lane 2). Amplification is evident by comparison of the unsonicated lanes with corresponding sonicated lanes. Results shown are representative of several independent experiments. Panels C and F show quantitation of PrPres as integrated intensity, i.e. the quantitative measure of protein detected by the scanner, from each lane of experiments shown in A, B, D, and E (using LyCor Odyssey scanner and software). Closed circles represent values for sonicated samples minus values for unsonicated freeze-control samples. Groups were compared by a 2-tailed nonparametric Mann Whitney test. For the 22L seed, the 170N and 170S substrates were significantly different (P<0.01), and for the RML seed, the 170N and 170S were not significantly different (P = 0.093).</p

Immunoblot analysis of PrPres in scrapie-infected mice.

<p>(A) Panel A shows immunoblots of brain homogenates tested using both 0.5mg and 0.25mg brain equivalents per lane. PrPres levels in clinical Tg330 mice (#255 and #019) were slightly lower than levels seen in clinical C57BL/10 mice. All C57BL/10 and Tg330 mice inoculated with 22L and ME7 scrapie were positive for PrPres by western blot at the time of clinical disease. In panels B and C, lanes 1 and 2 had 0.5 mg brain equivalents and other lanes had 1.0mg. (B) RML (stock 06) inoculations. Of fifteen transgenic mice tested, two (#573 and 778) were positive for PrPres at 451 and 518 days respectively. Four of the 13 PrPres-negative mice are also shown. RML (stock 81) inoculation of 14 mice resulted in none positive for PrPres by immunoblot (not shown). (C) 79A inoculations. Of fourteen transgenic mice tested, two mice (#657 and 470) were positive for PrPres at 439 and 545 dpi respectively. Four of the 12 PrPres-negative mice are also shown.</p