Figure 1.
Decreased Sho protein levels in the brain during experimental and natural prion disease.
(A) Western blot analysis of Sho protein levels in the brains of clinically ill wt CD-1 mice and meadow voles infected with RML prions. Sho levels were notably reduced in prion-infected brains compared to the uninfected brains, as probed by two distinct anti-Sho antibodies (the N-terminal antibody 06rSH-1 and the C-terminal antibody 06SH-3a). Prion disease is indicated by the presence of PK-resistant PrP in infected brains. (B) Decreased Sho levels in the brains of three sheep with natural scrapie as well as a sheep inoculated with the CH1641 scrapie strain compared to brains from healthy control animals. (C) In Tg(NSE-MoPrP) mice, PrPC expression is under the control of the neuron specific enolase (NSE) promoter and restricted to neurons. RML prion-infected Tg(NSE-MoPrP) mice exhibited diminished Sho protein levels compared to uninfected mice. (D) Sprn mRNA levels in wt FVB (black) and C57BL/6 (gray) mice (log2 change compared to mice inoculated with uninfected brain homogenate) remained relatively constant after infection with RML prions, arguing that the depletion of Sho protein levels during prion disease is a post-transcriptional process. Sprn mRNA data was extracted from the Prion Disease Database [26]. For Western blots, actin levels are shown as controls. PrP was probed using the antibodies HuM-D18 (A, C) or HuM-P (B). Molecular masses based on the migration of protein standards are shown in kilodaltons.
Figure 2.
Inverse relationship between Sho and PrPSc levels during prion disease in mice.
(A) Western blotting of Sho and protease-resistant PrPSc in brain homogenates prepared from wt mice infected with RML prions at the indicated days postinoculation (dpi). As Sho signals began to decrease, protease-resistant PrPSc increased. Actin levels are shown as a control. Molecular masses based on the migration of protein standards are shown in kilodaltons. Sho and PrP were probed using the antibodies 06rSH-1 and HuM-P, respectively. (B) Quantification of relative Sho (black) and PK-resistant PrPSc (gray) levels in RML-infected mice at the indicated days postinoculation. The inflection points for Sho reduction and PrPSc accumulation both occurred at ∼70 dpi. (C) Correlation analysis of Sho and PK-resistant PrPSc levels in the brains of RML-infected mice (n = 29). A significant, inverse correlation (P<0.0001; R2 = 0.85) was observed, indicating that increased protease-resistant PrPSc levels are associated with decreased Sho levels in the brain.
Figure 3.
Decreased Sho levels in different animal models infected with diverse prion strains.
(A) Western blot analysis of brain homogenates prepared from wt FVB mice (expressing the PrP-A allotype) infected with RML, 22L, Me7, and 301V prions, and B6.I mice (expressing the PrP-B allotype) infected with 87V and 301V prions. All inoculated mice developed prion disease, as indicated by the presence of protease-resistant PrPSc, and showed decreased Sho levels. (B) Western blot analysis of brain homogenates from hamsters infected with Sc237, 139H, HY and DY prion strains. All inoculated hamsters developed prion disease, as indicated by the presence of protease-resistant PrPSc, and showed depleted Sho levels. (C) In Tg(OvPrP) infected with scrapie SSBP prions and Tg(ElkPrP) mice infected with elk CWD prions, Sho levels were decreased compared to age-matched, uninfected animals. In addition to decreased Sho levels, clinically ill animals showed protease-resistant PrPSc in their brains. Wild-type mice and Tg mice overexpressing mouse PrP are shown for comparison. (D) Western blot analysis of brain homogenates prepared from Tg(HuPrP,M129) and Tg(HuPrP,V129) mice infected with human sCJD(MM1) and sCJD(VV2) prions, respectively. Clinically ill mice showed reduced levels of Sho compared to uninfected controls. For all panels, actin levels are shown as a control. Sho was probed with the antibody 06rSH-1; PrP was detected using antibodies HuM-D18 (A); 3F4 (B, D); or HuM-P (C), respectively. Molecular masses based on the migration of protein standards are shown in kilodaltons.
Figure 4.
Decreased Sho levels in ScN2a-Sho cells.
(A) Western blot analysis of Sho levels in untransfected N2a-Sho cells. Samples were treated with PNGaseF to remove N-glycans, as indicated. Blots were probed with anti-Sho antibodies 06rSH-1 (top blot) and 06SH-3a (bottom blot) recognizing N-terminal and C-terminal Sho epitopes, respectively. An asterisk (*) denotes a cross-reactive band of ∼17 kDa, which is also detected in N2a cells, recognized by the 06rSH-1 antibody. Whereas both the N- and C-terminal antibodies recognize full-length, unglycosylated Sho (∼16 kDa), the C-terminal antibody also detects an endoproteolytic Sho fragment (ShoC1 fragment). Molecular masses based on the migration of protein standards are shown in kilodaltons. (B) In heterogeneous ScN2a-Sho cells, Sho levels were not decreased. However, upon further subcloning of ScN2a-Sho cells to obtain a more uniform population of infected cells (ScN2a-Sho-1 and ScN2a-Sho-2 subclones), a notable decrease in Sho levels was observed. ScN2a-Sho cells harbor PK-resistant PrPSc, as detected by the antibody HuM-D18. Actin levels are shown as a control. Molecular masses based on the migration of protein standards are shown in kilodaltons. (C) Quantification of Sho levels in ScN2a-Sho-1 (n = 10) and ScN2a-Sho-2 (n = 5) subclones revealed a significant decrease of 40–45% compared to the uninfected parental cell line (n = 15) (***P<0.001).
Figure 5.
Unchanged Sho levels in mice with other neurodegenerative illnesses.
(A) Levels of Sho in the brains of Tg(APP23) and Tg(CRND8) mice, two Tg mouse models of Alzheimer's disease, were unaltered despite the high levels of cerebral Aβ present in aged mice. Actin and amyloid precursor protein (APP) levels are shown as controls. For comparison, Sho, APP, and Aβ levels in the brain of a wt FVB mouse are shown. Sho was probed with the antibody 06rSH-1; Aβ detected with the antibody 6E10, and APP with the antibody APPCT. (B) No change in Sho levels in either of two lines of Tg mice with a neurodegenerative disease caused by expression of mutant MoPrP(P101L). The brains of these mice have abundant levels of protease-sensitive, PTA-precipitable PrP but do not have any PK-resistant PrP. Sho and protease-resistant PrPSc in wt FVB mice infected with RML prions are shown for comparison. Sho was detected with the antibody 06rSH-1 and PrP was probed with the antibody HuM-D18. Actin levels are shown as a control. (C) Quantification of Sho levels in Tg(MoPrP,P101L) mice reveal only small decreases compared to wt mice (n = 3 for each group). In contrast, Sho levels in wt mice infected with RML prions are decreased by ∼80% (***P<0.001) compared to uninfected, wt mice. (D) Levels of Sho in the brains of Tg4053 mice overexpressing MoPrP inoculated with the MoSP2 strain of protease-sensitive prions were similar to those in age-matched Tg4053 mice inoculated with PBS. Actin levels are shown as a control. The presence of protease-sensitive prions in the brains of MoSP2-infected Tg4053 mice was confirmed by their ability to seed the polymerization of recombinant PrP into amyloid as demonstrated by increased ThT fluorescence signals in RT-QuIC experiments. Sho was detected with the antibody 06rSH-1. For all Western blots, molecular masses based on the migration of protein standards are shown in kilodaltons.
Figure 6.
Copurification of Sho and misfolded PrP from ScN2a-Sho-1 cell lysates.
Sho copurified with PrP molecules in lysates prepared from ScN2a-Sho-1 cells, but not with PrPC in N2a-Sho cells, as demonstrated by coimmunoprecipitation analyses. Nonspecific binding of misfolded PrP to the immunoprecipitation matrix was assessed by performing immunoprecipitations on ScN2a cells that do not express Sho and on ScN2a-Sho-1 cells in the absence of antibody (Mock). Samples were not treated with PK prior to Western blotting. Sho and PrP were probed using antibodies 06rSH-1 and HuM-D18, respectively. Molecular masses based on the migration of protein standards are shown in kilodaltons.
Table 1.
Incubation periods in Tg(Sho) mice following inoculation with different prion strains.
Figure 7.
Prion infection of transgenic mice overexpressing Sho.
(A) Western blot analysis of Sho levels in the brains of Tg24474 and Tg24488 mice that express mouse Sho at 12× and 20× levels, respectively, compared to wt FVB mice. Samples were treated with PNGaseF to remove N-glycans, as indicated. Sho was detected with two anti-Sho antibodies, one that recognizes the N-terminal region (06rSH-1, top blot) and the other recognizes C-terminal residues (06SH-3a, bottom blot). The C-terminal antibody detects an endoproteolytic fragment of Sho (ShoC1 fragment). An asterisk (*) denotes a cross-reactive band of ∼17 kDa, also observed in wt FVB mice, recognized by the N-terminal anti-Sho antibody. (B) ELISA-based quantification of PrPC levels in wt FVB mice; Tg24474 and Tg24488 mice overexpressing mouse Sho; and Tg3930 mice overexpressing human Sho demonstrated that PrPC levels were not significantly altered (P>0.05) in mice overexpressing Sho (n = 3 for each genotype). (C) Kaplan-Meier survival curves of wt and Tg(MoSho) mice infected with RML prions. No significant difference (P>0.05) was observed between the individual survival curves. (D) PrPC levels, PK-resistant PrPSc levels and glycosylation patterns after infection with RML prions were similar in Tg(MoSho) and wt FVB mice, as determined by Western blotting. The antibody HuM-P was used to detect PrP. For comparison, PrP in uninfected FVB and uninfected Tg24474 mice is shown. (E–J) Neuropathological analysis of RML-infected wt and Tg(MoSho) mice. Hippocampal sections from RML-infected wt (E, H); Tg24474 (F, I); or Tg24488 (G, J) mice were either stained with haematoxylin and eosin (E–G) or with the anti-PrP antibody HuM-P (H–J). Changes associated with prion disease, including spongiform degeneration (yellow arrows in panels E–G) and PrP deposition (brown staining in panels H–J), were apparent in all sections. No neuropathological differences were evident between RML-infected wt and Tg(MoSho) mice. Scale bar in panel E represents 100 µm and applies to all micrographs. CA1, CA1 pyramidal cell layer; cc, corpus callosum.
Figure 8.
Sho levels in prion-infected Tg(MoSho) and Tg(HuSho) mice.
(A) Levels of Sho were decreased in RML prion-infected Tg(MoSho) mice compared to uninfected controls. For comparison, Sho and protease-resistant PrPSc levels in RML-infected, wt FVB mice are shown. The antibody HuM-P was used to probe PrP, and the antibody 06rSH-1 used to detect Sho. Actin levels are shown as a control. (B) RML prion infection resulted in decreased levels of both full-length and endoproteolytically trimmed Sho in Tg(MoSho) mice. All samples were treated with PNGaseF. The 06SH-3a antibody recognizing a C-terminal Sho epitope was used. (C) Quantification of Sho levels in wt and Tg(MoSho) mice (n = 3 for each group) following infection with RML prions. In all infected mice, Sho levels decreased by ∼70% compared to uninfected mice. (D) Levels of Sho were decreased in RML prion-infected Tg(HuSho) mice compared to uninfected controls. The antibody HuM-P was used to probe PrP, and the antibody S-12 used to detect Sho. Actin levels are shown as a control. For all Western blots, molecular masses based on the migration of protein standards are shown in kilodaltons.
Figure 9.
Sho levels in prion-infected Tg mice expressing truncated or anchorless PrP.
(A) Western blots of brain homogenates from wt mice and Tg9949 mice (ΔN), which express MoPrP lacking residues 23–88, after infection with RML, 22L, and 301V prions. Despite developing prion disease and showing protease-resistant PrPSc in their brains, Tg9949 mice showed only slight reductions in Sho levels compared to wt mice following prion infection. Actin levels are shown as a control. (B) Quantification of Sho levels in wt and Tg9949 mice before and after infection with RML and 301V prions. Sho levels were reduced by ∼30% in prion-infected Tg9949 mice compared to the 65–75% reduction observed in infected, wt mice; this difference was statistically significant for both RML and 301V prions (***P<0.001, n = 3 for each condition). (C) Western blots of brain homogenates from wt mice and Tg(PrP-ΔGPI) mice, which express GPI-anchorless MoPrP, after infection with RML prions. Sho levels were lower in infected Tg(PrP-ΔGPI) mice harboring protease-resistant PrPSc in their brains compared to uninfected mice. (D) Quantification of Sho levels in wt and Tg(PrP-ΔGPI) mice after RML inoculation. Sho levels were decreased by ∼45% in prion-infected Tg(PrP-ΔGPI) mice compared to the ∼75% reduction observed in infected, wt mice (***P<0.001, **P<0.01, n = 3 for each condition). For the Western blots, Sho and PrP were probed with the antibodies 06rSH-1 and HuM-P, respectively. Molecular masses based on the migration of protein standards are shown in kilodaltons.
Figure 10.
Decreased Sho levels correlate with the amount of PrPSc C2 fragment present in prion-infected animals.
(A) Quantification of Sho levels in meadow voles before and after infection with RML, Sc237, and 301V prions. Sho levels were reduced by ∼90% in RML-infected voles compared to the ∼80% reduction observed in Sc237- and 301V-infected voles (*P<0.05 as determined by one-way ANOVA, n = 3–4 for each group). (B) Western blot analysis of Sho levels in the brains of prion-infected meadow voles. Infection with RML prions resulted in the largest decrease in Sho levels and the highest amount of PrPSc C2 fragment (determined after digestion with thermolysin (TL) and PNGaseF). The presence of PK-resistant PrPSc indicates prion disease. (C) Correlation analysis of Sho and relative PrPSc C2 fragment levels in the brains of prion-infected meadow voles (n = 11). A significant, inverse correlation (P<0.001) was observed, indicating that increased production of the PrPSc C2 fragment is associated with decreased Sho levels in the brain. (D) Western blot analysis of Sho levels in the brains of Tg(MoSho)24474 mice infected with RML, Me7, and 301V prions. The largest decrease in Sho levels was observed with RML and Me7 infections, which also resulted in the largest amounts of PrPSc C2 fragment (determined after digestion with TL and PNGaseF). In comparison, infection with 301V prions resulted in the smallest reduction in Sho levels and the lowest relative level of the PrPSc C2 fragment. Sho and PrP were probed using the antibodies 06rSH-1 and HuM-P, respectively. Actin levels are shown for comparison. Molecular masses based on the migration of protein standards are shown in kilodaltons.