We therefore tested whether Δ23–31 PrP was defective in endocytosis. N2a cells expressing either WT or Δ23–31 PrP were incubated on ice with an anti-PrP antibody, and then warmed to 37°C to initiate endocytosis. After this, cells were incubated in the absence () or presence () of PIPLC, which cleaves off any PrP remaining on the cell surface. Cells were then fixed, permeabilized, and incubated with a fluorescently labeled secondary antibody. Both WT and Δ23–31 PrP-expressing cells showed staining in the absence of PIPLC (). After PIPLC treatment, cells expressing WT PrP displayed a punctate pattern of intracellular staining, corresponding to endocytic structures containing PrP (). In contrast, Δ23–31 PrP cells lacked these structures (). These results indicate that Δ23–31 PrP is defective in endocytosis, confirming previous evidence regarding the role of residues 23–31 in this process.
Δ23–31 PrP converts into Δ23–31 PrP Sc in cultured cells
To test the conversion capability of Δ23–31 PrP in cell culture, scrapie-infected N2a cells were transiently transfected with plasmids encoding epitope-tagged WT or Δ23–31 PrP. Once confluent, cells were collected, a portion of the cell lysate was digested with PK, and both digested and undigested lysates were analyzed by Western blotting using 3F4 antibody to detect epitope-tagged PrP. Δ23–31 PrP was converted into PK-resistant forms that comigrated with those generated from WT PrP (, lanes 5, 6), indicating that deletion of the polybasic domain does not abolish the conversion of PrPC into PrPSc. However, we noted that, in proportion to the amount of undigested PrP, the deletion mutant generated less protease-resistant protein than did its WT counterpart.
Generation of transgenic mice expressing Δ23–31 PrP
To analyze the role of residues 23–31 in prion propagation and toxicity in vivo
, we generated Tg mice expressing Δ23–31 PrP under the control of the PrP half-genomic promoter (Borchelt et al., 1996
). Tg founders were bred to Prn-p0/0
mice on the C57BL/6J background, and the expression level for each Tg line was then quantitated by Western blotting. Three different Tg(Δ23–31) mouse lines were selected for further study, expressing approximately six times, four times, and one times the endogenous WT PrP level (, lanes 3–5). These relative expression levels were confirmed by quantitative Western blotting and analysis by Storm and Odyssey imaging systems (data not shown). Δ23–31 PrP from each of the Tg lines displayed several glycoforms, with the diglycosylated form being predominant (, lanes 1–5). After deglycosylation with PNGase F, all PrP forms collapsed into a single band migrating at ~24 kDa, slightly smaller than the corresponding band for WT PrP in Tga20+/+
and nontransgenic C57BL/6J (referred to as non-Tg) mice (, lanes 6 –10).
Figure 3 Expression and solubility of Δ23–31 PrP in transgenic mice. A, Equal volumes of protein from the brains of mice of the indicated genotypes were either untreated (lanes 1–5) or were treated with PNGase F (lanes 6 –10) before (more ...)
We also examined the localization and solubility of Δ23–31 PrP from transgenic mice. Like WT, Δ23–31 PrP was expressed on the plasma membrane of cerebellar granular neurons (Turnbaugh et al., 2011
). In addition, both WT and Δ23–31 PrPs were soluble in detergents (, lanes 3– 6), in contrast to an aggregation-prone mutant (PG14) that was partially detergent insoluble (, lanes 7, 8). Together, these results show that Δ23–31 and WT PrPs have similar localization and biochemical properties in vivo
Tg(Δ23–31) mice show prolonged survival after scrapie inoculation
Mice expressing Δ23–31 PrP at six times, four times, or one times on the Prn-p
background showed no evidence of spontaneous disease and remained healthy for >600 d (data not shown). To test the role of residues 23–31 in prion conversion in vivo
, we inoculated Tg(Δ23–31 1×
), Tg(Δ23–31 4×
), Tg(Δ23–31 6×
) mice on the Prn-p
background with the RML strain of scrapie and compared survival times as well as accumulation of PK-resistant PrP in the brains of these animals. As controls, we also inoculated three kinds of mice with WT PrP expression levels spanning those of mutant PrP in the TgΔ23–31 lines: non-Tg mice, which express endogenous PrP (1 times); and Tga20 +/0
and Tga20 +/+
mice, which express WT PrP from a transgene at 5 and 10 times, respectively (Shmerling et al., 1998
Surprisingly, none of the nine Tg(Δ23–31 1×) mice displayed symptoms of disease at >400 d postinoculation (dpi) (, green line; ). In contrast, non-Tg control mice became terminally ill at ~160 dpi (, gray line). Both Tg(Δ23–31 4×) (, blue line) and Tg(Δ23–31 6×) mice (, red line) also showed a significant increase in life span, compared with Tga20 +/0 and Tga20 +/+ overexpressing controls (, dashed black and solid black lines, respectively). Interestingly, Tg(Δ23–31 4×) mice fell into two distinct groups, with one group surviving up to 166 dpi, and the second reaching terminal disease much later, between 336 and 427 dpi (, blue line; ). Tg(Δ23–31 6×) mice had a more homogenous survival time, with all mice becoming terminally ill at an average of ~130 dpi (, red line; ). Tga20 +/+ and Tga20 +/0 control mice had much shorter survival times than any of the Tg(Δ23–31) mice, succumbing at 73 and 81 dpi on average, respectively (, solid black and dashed black lines; ).
Figure 4 Tg(Δ23–31) mice survive longer than control mice after scrapie inoculation. Survival times were monitored in RML-inoculated mice of the following genotypes: Tga20 +/+ (solid black line); Tga20 +/0 (dashed black line); non-Tg (gray line); (more ...)
Tg(Δ23–31) mice have increased survival times after scrapie inoculation
These results show that deletion of PrP residues 23–31 dramatically extends the life span of Tg mice inoculated with RML prions, suggesting that these residues play an important role in the process of prion propagation and/or toxicity.
Terminally ill Tg(Δ23–31) mice accumulate low levels of PK-resistant PrP
To determine whether the increase in life span observed in RML-injected Tg(Δ23–31) mice was related to a decreased accumulation of PrP Sc, brain homogenates from mice at 70 dpi and from terminally ill mice were digested with PK, and the amount of PK-resistant PrP was analyzed by Western blot (, ). At 70 dpi, no PK-resistant PrP was detected in RML-injected Tg(Δ23–31) mice (, lanes 7–12), while WT-expressing controls accumulated low levels (, lanes 1– 6). At the time of terminal disease, when control mice showed prominent accumulation of PrPSc, Tg(Δ23–31) mice had little or no PK-resistant PrP (). In particular, asymptomatic Tg(Δ23–311×) mice, killed at ~300 dpi, showed no detectable, PK-resistant PrP in their brains (, lanes 15, 16), while, as expected, non-Tg mice had high levels (, lanes 5, 6). In addition, the average amount of PK-resistant PrP found in Tg(Δ23–316×) and short-survival Tg(Δ23–314×) mouse brains was only 10 and 16% of the level of non-Tg mice (, lanes 7–14), while Tga20+/+ and Tga20+/0 mice accumulated substantially more PK-resistant PrP (46 and 62% the level of non-Tg animals, respectively; , lanes 1– 4). Further evaluation of the brains of long-survival Tg(Δ23–314×) mice revealed that these animals accumulated higher levels of PK-resistant PrP (, lanes 8 –11) than short-survival mice of the same genotype (, lanes 4 –7). However, even the long-survival mice had only 51% of the amount of PK-resistant PrP found in the brains of terminally ill Tga20+/0 mice (, lanes 1–2).
Figure 5 Tg(Δ23–31) mice accumulate less PrP Sc than controls at 70 dpi. Brain homogenates from RML-inoculated mice of the indicated genotypes at 70 dpi were treated without (top panel) or with (bottom panel) PK and were then subjected to Western (more ...)
Figure 6 Tg(Δ23–31) mice accumulate greatly reduced amounts of PrP Sc. A, Brain homogenates from RML-inoculated mice of the indicated genotypes at the terminal stage were treated without (top panels) or with (bottom panels) PK and were then subjected (more ...)
To test the possibility that Tg(Δ23–31) mice accumulate a form of PrP Sc that is protease sensitive, brains from terminally ill Tg(Δ23–31 6×) mice were digested with increasing concentrations of PK. At all concentrations of PK, the amount of PK-resistant PrP in the brain of Tg(Δ23–31 6×) mice was much lower than that of Tga20 mice (, top panels). However, a longer exposure of the Western blots (, bottom panels) revealed that the small amount of PrPSc formed in Tg(Δ23–316×) mice was fully resistant to PK (up to 50 μg/ml). Collectively, these results demonstrate that RML-inoculated Tg(Δ23–31) mice accumulate much less PK-resistant PrP over the course of disease than mice expressing similar levels of the WT protein. However, the small amount of PrP Sc present in the brains of Tg(Δ23–31) mice is still fully PK resistant, suggesting that this biochemical property of the RML scrapie strain is preserved when it is passaged into Tg(Δ23–31) mice.
Primary sites of PrP Sc accumulation and spongiform degeneration are similar in RML-injected Tg(Δ23–31) and control mice
One possible explanation for the extended life span observed in PrP Sc-infected Tg(Δ23–31) mice is that a new strain, with neuropathological properties different from the original inoculum, was generated after passaging RML prions into these mice. To address this possibility, the distribution of PrP Sc and the presence of spongiform degeneration were assessed, comparing terminally ill, RML-infected Tg(Δ23–31) and control mice (). The distribution of PrPSc in brain sections was assessed by immunohistochemical staining, and the presence of spongiform degeneration was evaluated by hematoxylin and eosin staining. RML-inoculated Tga20+/+, Tga20+/0, and non-Tg control mice accumulated PrPSc primarily in the thalamus () and brainstem (), and to a much lesser extent in the cortex () and other brain areas (data not shown). Spongiform degeneration in these animals was detected primarily in the brainstem (). As expected, the amount of PrPSc detected in the different brain areas of RML-injected Tg(Δ23–314×) and Tg(Δ23–316×) mice was generally lower than that of controls. However, both of these mouse lines showed a pattern of PrPSc distribution identical with that of WT-expressing controls, with the thalamus () and brainstem () being the main sites of PrPSc accumulation, while almost no PrPSc was detected in the cortex () and other brain areas (data not shown). Moreover, spongiform degeneration was observed mainly in the brainstem of Tg(Δ23–314×) and Tg(Δ23–316×) mice (). PrPSc deposition and spongiform change were comparable in Tg(Δ23–314×) mice with short and long survival times (data not shown). Importantly, no PrPSc staining or spongiosis was detected in brain sections from RML-injected, Tg(Δ23–311×) mice at 300 dpi (), or in uninoculated Tga20+/+ mice () and Tg(Δ23–316×) mice ().
Figure 7 Tg(Δ23–31) and control mice display PrP Sc accumulation and spongiform change in similar areas of the brain. A1–C8, Paraffin sections from mice of the indicated genotypes were stained for PrP Sc. Representative staining of the (more ...)
To confirm that the overall histopathological profile was similar in RML-inoculated Δ23–31 PrP and WT-expressing mice, we scored the extent of PrP Sc
deposition and spongiosis that occurred in each of six different brain regions (). All terminally ill Tga20 +/+
, Tga20 +/0
, Tg(Δ23–31 6×
), and Tg(Δ23–31 4×
) mice showed similar patterns of PrP Sc
deposition, with accumulation primarily in the thalamus and brainstem and the greatest amount of spongiosis in the brainstem. As expected based on previous work (Karapetyan et al., 2009
), non-Tg mice showed more extensive PrP Sc
deposition throughout the brain, and higher levels of spongiform change in the cerebellum.
Quantitation of regional histopathology profiles for RML-inoculated mice
In summary, the accumulation of PrP Sc in the thalamus and brainstem, as well as the presence of spongiform degeneration of the brainstem, both typical of the RML strain, were indistinguishable between WT-expressing controls and Tg(Δ23–31 4×) or Tg(Δ23–31 6×) mice, although both of these neuropathological features were generally less severe in mice expressing the PrP mutant. These results argue against the possibility that the extended life span of RML-inoculated Tg(Δ23–31) mice is related to generation of a new prion strain displaying different properties than the original inoculum.
Deletion of residues 23–31 does not create a sequence barrier for prion propagation
Another possible explanation for the longer survival times observed in RML-infected Tg(Δ23–31) mice is the presence of a sequence mismatch between the original RML seed (which carries a WT PrP sequence) and the Δ23–31 PrP substrate. This mismatch could constitute a barrier for prion propagation and lead to suboptimal conversion of Δ23–31 PrP into Δ23–31 PrP Sc. To test this possibility, we performed secondary passage experiments, inoculating Tg(Δ23–31 6×), Tga20 +/+, and non-Tg hosts with brain homogenates from RML scrapie-infected Tg(Δ23–31 6×) mice (hereafter referred to as RML Δ23–31). If a sequence mismatch were responsible for the prolonged survival times seen in the primary inoculation experiments, then secondary passage of RML Δ23–31 into Tg(Δ23–31 6×) host mice should result in shorter survival. In fact, we observed no difference in the life span of Tg(Δ23–31 6×) mice infected with the original, WT RML inoculum compared with those inoculated with RML Δ23–31 (, red lines with circles and squares, respectively; selected data from and are reported again in and to allow direct comparison). Moreover, the survival times after inoculation of Tga20 +/+ mice with WT RML and RML Δ23–31 were almost identical (, black lines with circles and squares, respectively; ). The only statistically significant difference was found between non-Tg mice inoculated with RML and RML Δ23–31, with the latter surviving an additional 10 dpi, on average (, gray lines with circles and squares, respectively; ). This small increase in life span may be attributable to the lower amount of PK-resistant PrP Sc in the RML Δ23–31 inoculum, compared with the WT RML inoculum (as shown in ).
Figure 8 Secondary passage of RML Δ23–31 scrapie into Tg(Δ23–31) mice does not shorten survival times. A, Survival times were monitored in mice of the following genotypes inoculated with RML or RML Δ23–31: Tg(Δ23–31 (more ...)
Mice inoculated with RML or RML Δ23–31 show similar survival times
Together, these data argue against the idea that deletion of residues 23–31 from PrP C creates a sequence barrier that affects propagation of the RML strain.
RML Δ23–31 maintains the biological properties of the original inoculum
To test whether the biochemical and neuropathological properties of the original RML inoculum were maintained after passaging this strain into Tg(Δ23–31 6×) mice, we compared the amount and site of accumulation of PK-resistant PrP in mice infected with RML or with RML Δ23–31. Brain homogenates from terminally ill Tg(Δ23–31 6×), Tga20 +/+, or non-Tg mice inoculated with RML or with RML Δ23–31 were incubated with PK and analyzed by Western blotting (). We observed low or undetectable levels of PK-resistant PrP in the brains of Tg(Δ23–31 6×) mice inoculated with either RML (, lanes 2, 3) or RML Δ23–31 (, lanes 4 –7). Tga20 +/+ and non-Tg mice consistently accumulated larger amounts of PK-resistant PrP, which was similar after infection with either RML (, lane 1) (see also , lanes 1, 2, and 5, 6) or RML Δ23–31 (, lanes 8 –11 and 12–15).
We then assessed the distribution of PrPSc as well as the presence of spongiform degeneration in different brain areas from terminally ill Tg(Δ23–31) and control mice infected with RMLΔ23–31 (, ). Only low levels of PrPSc were detected in RMLΔ23–31-infected Tg(Δ23–316×) mice, although, as for the first passage, the main sites of accumulation were the thalamus () and brainstem (). The same two areas were also found to be the primary sites of PrPSc accumulation in RMLΔ23–31-infected Tga20+/+() and non-Tg () mice. All the animals showed a similar pattern of spongiform degeneration, with the brainstem being the primary site affected ().
Figure 9 RML strain characteristics are preserved during secondary passage in Tg(Δ23–31) mice. Paraffin sections from terminally ill Tga20 +/+, non-Tg, and Tg(Δ23–31 6×) mice inoculated with RML Δ23–31 display (more ...)
Quantitation of regional histopathology profiles for RML Δ23–31-inoculated mice
To further analyze the extent and location of PrP Sc deposition and spongiosis in the brains of RML Δ23–31-infected mice, we quantified each of these pathologies in the cortex, hippocampus, cerebellum, brainstem, thalamus, and striatum of terminally ill mice of each genotype (). The highest levels of PrP Sc were localized in the brainstem and thalamus of Tga20 +/+ and Tg(Δ23–31 6×) mice, and the most severe spongiform degeneration was observed in the brainstem. As expected, non-Tg mice showed a wider extent of PrP Sc deposition, as well as increased spongiosis in the cerebellum.
These results demonstrate that both the biochemical and neuropathological properties of the RML inoculum are unaltered after passaging this strain into Δ23–31 PrP-expressing mice.
Deletion of residues 23–31 makes PrP an inefficient substrate, but does not compromise its seeding ability
To gain additional insights into the role of residues 23–31 in prion conversion, we undertook experiments using an in vitro conversion system to complement our in vivo studies in mice. Protein misfolding cyclic amplification (PMCA) was used to test whether the original RML inoculum was able to seed the misfolding of WT or Δ23–31 PrP. Brain homogenates from either Tga20 +/0 or Tg(Δ23–31 6×) mice were used as substrates for the reaction. The RML inoculum efficiently seeded the misfolding of full-length PrP (, top right panel) but not Δ23–31 PrP (, bottom right panel). These results indicated that, as observed in vivo, PrP molecules deleted for residues 23–31 are inefficiently converted into PrP Sc.
Figure 10 Deletion of residues 23–31 makes PrP an inefficient substrate, but does not compromise its seeding ability. PMCA reactions were run using the indicated seeds and substrates. Experiments were performed at least three times, and a representative (more ...)
Next, we compared the seeding activity of RML scrapie passaged into Tga20 +/0 or Tg(Δ23–31 6×) mice. To allow direct comparison of the seeding activity of the two inocula, similar amounts of PK-resistant RML or RML Δ23–31 were used to seed the misfolding of WT PrP derived from non-Tg brain homogenates (, left panels). Consistent with our secondary passage experiments in Tga20 +/+ mice (), we found that RML and RML Δ23–31 seeds were equally capable of inducing the conversion of full-length PrP (, right panels), indicating that the deletion of residues 23–31 does not affect the seeding activity of PrP Sc.
These in vitro results recapitulated the observations made in vivo, demonstrating that deletion of residues 23–31 from PrP C impairs its conversion into PrP Sc. However, once conversion is established, the resulting RML Δ23–31 molecules show the same seeding activity as the original inoculum.
PMCA detects infectivity in healthy Tg(Δ23–31 1×) mice
As described above, RML-infected Tg(Δ23–31 1×) mice did not show any clinical or neuropathological signs of disease for >400 dpi. To test whether the brains of these mice accumulated subclinical amounts of infectious PrP Sc, we took advantage of the high sensitivity of the PMCA reaction. Surprisingly, we found that brain homogenates from healthy, RML-infected Tg(Δ23–31 1×) mice at 300 dpi were able to seed the misfolding of WT PrP derived from Tga20 +/+ mice (, right panel) in the absence of any detectable PK-resistant seed (, left panel). Additionally, when brain homogenates from the same Tg(Δ23–31 1×) mice were used to inoculate Tga20 +/+ indicator mice, these mice developed prion disease (data not shown).
These results suggest that Tg(Δ23–31 1×) mice accumulated either very low levels of PK-resistant PrP Sc or a PK-sensitive, nonpathogenic form of PrP Sc that was only detectable using high-sensitivity techniques such as passage into PrP-overexpressing mice or PMCA.
Deletion of residues 23–31 compromises binding of PrP C to PrP Sc
To test whether inefficient conversion of Δ23–31 PrP is a consequence of decreased binding to PrP Sc, we performed immunoprecipitation experiments comparing the amount of PrP Sc pulled down by WT or Δ23–28 PrP molecules (the latter, like Δ23–31 PrP, harbors a deletion of the polybasic domain). Recombinant (rec), myc-tagged WT or Δ23–28 PrP was incubated with brain homogenates from RML-infected, non-Tg mice. An anti-myc antibody was then used to immunoprecipitate the PrP C–PrP Sc complexes, followed by digestion with PK to detect PrP Sc. Detection of myc-tagged PrP in absence of PK treatment confirmed that both recWT and recΔ23–28 proteins were efficiently immunoprecipitated in presence or absence of PrP Sc (, top panel, lanes 2–5). Importantly, we found that recΔ23–28 PrP was much less efficient in pulling down PrP Sc when compared with the recWT PrP control (, bottom panel, compare lanes 2, 4). Averaging the results of three independent experiments, the polybasic domain mutant pulled down 41.8% (SEM, ±18.0) of the PrP Sc that was pulled down by WT PrP. No signal was detected when the immunoprecipitation was performed in absence of RML brain homogenate, recPrP, or anti-myc antibody (, bottom panel, lanes 3, 5, 6, 7). These results indicate that the impaired conversion of Δ23–31 PrP is likely due to defective binding to PrP Sc seeds.
Figure 11 The N-terminal, polybasic domain mediates binding between PrP C and PrP Sc. A, Myc-tagged WT or Δ23–28 recombinant PrPs were incubated in the presence or absence of RML-infected brain homogenate before immunoprecipitation with anti-myc (more ...)