Because prions are notoriously difficult to inactivate chemically and often acquire protease resistance [Prusiner 1982, recent updates covered in Peretz 2006], how they could possibly be degraded in vivo was a long-standing mystery [Aguzzi & Falsig 2012].
Meanwhile, with the discovery that abolishing PrP expression reverses prion disease in mice [Mallucci 2003] and the rise of depleting PrP as a potential therapeutic strategy, it now seems equally important to know how PrPC is degraded.
In a recent post, I got up to speed on the basics of protein degradation in the cell. In this post my goal is to review the evidence for the lysosomal role in PrP degradation. My next post will cover the proteasome.
lysosomal degradation of PrP promoted by autophagy-inducing drugs
A great deal of evidence for lysosomal degradation of PrP comes from the fact that several autophagy-inducing drugs have been found to reduce PrPC and/or PrPSc levels via hastened degradation – in cell culture and in some cases in vivo as well.
Some of the authors measured LC3-II [Mizushima & Yoshimori 2007 (ft), Tanida 2008], considered to be a marker of autophagy, in order to pinpoint the compounds’ mechanism of action. As noted previously, I find many authors use the term “autophagy” to refer not to a particular pathway, but rather to any lysosomal degradation of the cell’s own proteins. I believe this interpretation applies to LC3 assays as well. As I understand it, LC3 is a marker of the incorporation of cytosolic material into autophagosomes [Tanida 2008], while PrP is a cell-surface protein which must instead reach the lysosome by endocytosis and the subsequent fusion of endocytic vesicles with endosomes bound for lysosomes. Thus although LC3 may directly be part of a particular autophagic pathway, it seems that here it is acting as an indirect marker of overall lysosomal activity. Therefore in this context it seems the authors might do just as well to say they measured “lysosomal activity” rather than “autophagy”. If you know otherwise, please leave me a note.
Here is a brief summary of the compounds that have to date been reported to activate lysosomal degradation and thereby increase the rate of degradation of PrP.
- Imatinib (aka STI571) can, at high enough concentrations, completely abolish PrP-res in ScN2a cells but does not at all affect PrPC levels [Ertmer 2004 (ft)]. This effect was reduced by NH4Cl, a compound which inhibits lysosomal degradation by raising lysosomal pH, and therefore ascribed to increased lysosomal degradation. In vivo, imatinib was found to delay neuroinvasion and extend survival if administered early in peripheral prion infection, but did not inhibit prion replication in the brain and did not extend incubation times if administered after neuroinvasion [Yun 2007].
- Trehalose reduced PrP-res in ScN2a cells in a dose- and time-dependent manner, with a reduction of up to 80% in the best case [Aguib 2009 (ft)]. It had no effect on PrPC. Because trehalose affected LC3 and its antiprion effects were abolished by 3-MA (an autophagy-inhibiting compound), its effects were ascribed to autophagy. In peripherally infected mice, trehalose delayed prion accumulation in the spleen initially but had no longer-term effect on splenic PrPSc accumulation and ultimately did not affect survival.
- Lithium reduced PrPC levels slightly (20%) and PrP-res levels more dramatically (up to 90% for the longest treatment) in ScN2a cells [Heiseke 2009]. Lithium led to an increase in LC3-positive autophagic bodies in the cells, and its antiprion effects were reduced by 3-MA, so its effects were attributed to autophagy. It had no effect in vivo in intracerebrally infected animals.
- Rapamycin (aka Sirolimus) reduced PrP-res by about 60% in ScN2a cells [Heiseke 2009] though its effect on PrPC was not tested. It prolonged survival in intracerebrally infected mice by about 7% [Heiseke 2009] and 9% (at a high dose) in Tg(GSS) mice (where it also abolished PrP plaques) [Cortes 2012 (ft)], though it had no effect when started late in disease (at “clinical onset”, timepoint ~.88) [Mukherjee 2010].
- Tacrolimus (aka FK506, a “rapalogue” similar to rapamycin) is a delayed death by about 4% when administered late in disease (at “clinical onset”, timepoint ~.88) [Mukherjee 2010], a property attributed to its inhibition of calcineurin. More recently tacrolimus turned up in a screen for compounds that reduce PrPC expression, and its effects in cell culture (up to 70% reduction in PrPC) were attributed to reduced translation of PrP, while autophagy was ruled out as the drug did not affect LC3 [Karapetyan & Sferrazza 2013]. In that study, no effect was seen in vivo in mice treated only from 20-50 dpi (timepoints .13 – .34). However a new study last month did find that tacrolimus did indeed affect LC3 ratios and by implication was activating lysosomal activity [Nakagaki 2013 (ft)]. Nakagaki found an 11% extended survival in treated mice, but only when the drug was administered continuously from early on (20 dpi, timepoint ~.16).
For the sake of completeness, I’ll mention two other compounds which may be relevant. Amcinonide has been shown to reduce PrPC levels in cell culture through increased degradation [Poncet-Montange 2011], but this has not yet been specifically tied to lysosomal activity in any published report of which I am aware. Glucocorticoids (of which amcinonide is one) have often been reported to activate autophagy [e.g. Grander 2009, Xia 2010 - just Google and you'll find plenty more]. Resveratrol has also been reported to activate autophagy and reduce the toxicity of PrP 106-126 in cell culture [Jeong 2012], but to my knowledge its effects on full-length PrP degradation have not been reported.
Proteins in the (mouse) brain turn over slowly, with an average half life of 9 days [Price 2010]. In comparison, PrPC turns over quite rapidly, with a half life of 18 hours (again in the mouse brain) [Safar 2005 (ft)]. We know that PrPC cycles between the cell surface and endosomal compartments about once every hour [Harris 2003 (ft)], so it’s plausible that at least some of this degradation occurs when these endosomal compartments fuse with lysosomes, leading to lysosomal degradation of PrP. The fact that several different studies have found that autophagy-activating drugs are capable of reducing PrP levels provides strong evidence for a lysosomal role in PrP degradation.
Some of these studies found that autophagy inducers reduced PrPC while others did not, and the effect on PrPSc (when both were measured) was always greater. Unfortunately, none of these compounds had large effect sizes in vivo, and many had no effect at all in intracerebrally infected animals. Inducing autophagy is in no way specific to PrP, so it seems intuitively difficult to induce a therapeutically relevant amount of PrP reduction without also affecting other proteins to a toxic degree. Still, promoting lysosomal degradation of PrP could potentially be one piece of a cocktail solution to prion diseases.