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The Transcellular Spread of Cytosolic Amyloids, Prions, and Prionoids

2009; Cell Press; Volume: 64; Issue: 6 Linguagem: Inglês

10.1016/j.neuron.2009.12.016

1097-4199

Adriano Aguzzi, Lawrence Rajendran,

RNA regulation and disease

Recent reports indicate that a growing number of intracellular proteins are not only prone to pathological aggregation but can also be released and “infect” neighboring cells. Therefore, many complex diseases may obey a simple model of propagation where the penetration of seeds into hosts determines spatial spread and disease progression. We term these proteins prionoids, as they appear to infect their neighbors just like prions—but how can bulky protein aggregates be released from cells and how do they access other cells? The widespread existence of such prionoids raises unexpected issues that question our understanding of basic cell biology. Recent reports indicate that a growing number of intracellular proteins are not only prone to pathological aggregation but can also be released and “infect” neighboring cells. Therefore, many complex diseases may obey a simple model of propagation where the penetration of seeds into hosts determines spatial spread and disease progression. We term these proteins prionoids, as they appear to infect their neighbors just like prions—but how can bulky protein aggregates be released from cells and how do they access other cells? The widespread existence of such prionoids raises unexpected issues that question our understanding of basic cell biology. Imagine that you are a neuroscientist vacationing on Mars. One day you encounter a colony of Martians that, as it happens, look similar to water bottles. The Martians are highly distressed and seek your advice, as their community is plagued by an enigmatic transmissible disease. Intrigued, you agree to help. It turns out that the bodies of your exobiotic friends consist of bottles filled with a supersaturated salt solution. At some point crystals have started forming in one individual, and then crystallization has somehow been transferred to other community members. Lacking molecular insight, you would initially conclude that the Martians are affected by an infectious agent. Through ingenuity and technology, you may then discover that the infectious agent is exceedingly simple and homogeneous, that it lacks informational nucleic acids, and that it is generated both by ordered aggregation of an intrinsic precursor and by appositional growth of extrinsically added seeds. Your discovery will earn you the Intergalactic Nobel Prize, yet two crucial questions remain unanswered: how do the crystals transfer between individuals, and what can be done to prevent this from happening? Middle-aged readers may feel reminded of the plot for Andromeda Strain, a stunningly prescient novel published in 1969 by the late Michael Crichton. But the sci-fi scenario described above is also the blueprint of Prusiner's hypothesis of prion propagation. Over time, we have learned that prions consist of PrPSc, higher-order aggregates of a physiological protein termed PrPC. Accordingly, prions propagate through elongation and breakage of PrPSc aggregates (Aguzzi and Polymenidou, 2004Aguzzi A. Polymenidou M. Mammalian prion biology: one century of evolving concepts.Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar)—not unlike the crystals vexing our extraterrestrial friends. There is mounting evidence (Clavaguera et al., 2009Clavaguera F. Bolmont T. Crowther R.A. Abramowski D. Frank S. Probst A. Fraser G. Stalder A.K. Beibel M. Staufenbiel M. et al.Transmission and spreading of tauopathy in transgenic mouse brain.Nat. Cell Biol. 2009; 11: 909-913Crossref PubMed Scopus (1119) Google Scholar, Frost et al., 2009Frost B. Ollesch J. Wille H. Diamond M.I. Conformational diversity of wild-type Tau fibrils specified by templated conformation change.J. Biol. Chem. 2009; 284: 3546-3551Crossref PubMed Scopus (151) Google Scholar, Ren et al., 2009Ren P.H. Lauckner J.E. Kachirskaia I. Heuser J.E. Melki R. Kopito R.R. Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates.Nat. Cell Biol. 2009; 11: 219-225Crossref PubMed Scopus (326) Google Scholar, Desplats et al., 2009Desplats P. Lee H.J. Bae E.J. Patrick C. Rockenstein E. Crews L. Spencer B. Masliah E. Lee S.J. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein.Proc. Natl. Acad. Sci. USA. 2009; 106: 13010-13015Crossref PubMed Scopus (1011) Google Scholar, Luk et al., 2009Luk K.C. Song C. O'Brien P. Stieber A. Branch J.R. Brunden K.R. Trojanowski J.Q. Lee V.M. Exogenous {alpha}-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 20051-20056Crossref PubMed Scopus (545) Google Scholar) suggesting that the events sketched above, far from being confined to science-fiction and prion diseases (whose incidence in humans is just ≈1/106/year), may underlie highly prevalent human diseases of the brain and many other organs. The unifying characteristics of all these diseases is the aggregation of proteins into highly ordered stacks, henceforth termed “amyloids” irrespective of their size. Since PrPSc undoubtedly fulfills the latter definition of amyloid, one is led to wonder whether the prion principle may be much more pervasive than previously appreciated and whether many more diseases of unknown cause may eventually turn out to rely on prion-like propagation (Table 1, upper panel). Even more intriguingly, a number of proteins appear to exert normal functions when arranged in highly ordered stacks that are similar to amyloids and to prionoids (Table 1, lower panel).Table 1Potential Prionoids in Health and Disease (Adapted from Aguzzi, 2009Aguzzi A. Cell biology: Beyond the prion principle.Nature. 2009; 459: 924-925Crossref PubMed Scopus (153) Google Scholar)Phenotype/FunctionProteinMolecular TransmissibilityBona Fide InfectivityPrion diseasesPrPSc (luminal)yesyesAlzheimer's diseaseAβ (luminal)yesin APP-overexpressing miceTauopathiesTau (cytosolic)possiblynot shownParkinson's diseaseα-synuclein (cytosolic)host-to-graftnot shownAA amyloidosisSAA (luminal)yesprobableHuntington's diseasePolyQ (nuclear)yesnot shownSuppressed translational termination (yeast)Sup35yeslimitedBiofilm production (bacteria)bacterial curlinyesquestionableHeterkaryon incompatibility (fungi)Het-syeslimitedPituitary secretory granulespeptide hormonesnot shownnot shownMammalian skin pigmentationPmel17not shownnot shown Open table in a new tab There is one crucial difference between bona fide prion diseases and all other amyloids and prion-like phenomena hitherto described in uni- and pluricellular organisms (Table 1). Prions are infectious agents, transmissible between individuals, and tractable with microbiological techniques—including, e.g., titer determinations. Even if certain amyloids of yeast and mammals appear to infect neighboring molecules and sometimes neighboring cells, they do not propagate within communities, and none of them were found to cause macroepidemics such as Kuru and bovine spongiform encephalopathy. We have therefore termed these self-aggregating proteins “prionoids” (Aguzzi, 2009Aguzzi A. Cell biology: Beyond the prion principle.Nature. 2009; 459: 924-925Crossref PubMed Scopus (153) Google Scholar), since the lack of microbiological transmissibility precludes their classification as true prions. Some prionoids may soon qualify for an upgrade to prion status. At least in select settings, amyloid A (AA) amyloidosis may exist as a truly infectious disease based on a self-propagating protein. AA amyloid consists of orderly aggregated fragments of SAA protein, whose deposition can damage many organs of the body. Somewhat bizarrely, AA aggregation is also present in the liver of force-fed geese, hence contributing to the pathophysiology of foie gras (Solomon et al., 2007Solomon A. Richey T. Murphy C.L. Weiss D.T. Wall J.S. Westermark G.T. Westermark P. Amyloidogenic potential of foie gras.Proc. Natl. Acad. Sci. USA. 2007; 104: 10998-11001Crossref PubMed Scopus (83) Google Scholar). AA seeds can induce amyloidosis upon transfer of white blood cells (Sponarova et al., 2008Sponarova J. Nyström S.N. Westermark G.T. AA-amyloidosis can be transferred by peripheral blood monocytes.PLoS ONE. 2008; 3: e3308Crossref PubMed Scopus (39) Google Scholar). Furthermore, AA seeds are excreted with the feces, and AA amyloidosis is endemic in populations of cheetah (Zhang et al., 2008Zhang B. Une Y. Fu X. Yan J. Ge F. Yao J. Sawashita J. Mori M. Tomozawa H. Kametani F. Higuchi K. Fecal transmission of AA amyloidosis in the cheetah contributes to high incidence of disease.Proc. Natl. Acad. Sci. USA. 2008; 105: 7263-7268Crossref PubMed Scopus (88) Google Scholar). It is therefore tantalizing to suspect that amyloid may entertain the complete life cycle of an infectious agent, including transmission by the orofecal and hematogenous route—similarly to enteroviruses and, perhaps, scrapie prions. While there may be many other good reasons to avoid foie gras, including, e.g., animal welfare concerns, gourmets may not need to panic: under experimental conditions, AA amyloidosis is only transmitted to AgNO3-pretreated mice that display elevated levels of the SAA precursor protein. Alzheimer's disease (AD) has long been suspected to be a transmissible disease, but these suspicions have never materialized in epidemiological studies. On the other hand, Mathias Jucker and Lary Walker observed that injection of the Aβ peptide from human AD brains induced robust and convincing aggregation of Aβ in transgenic mice overexpressing the Aβ precursor protein, APP (Kane et al., 2000Kane M.D. Lipinski W.J. Callahan M.J. Bian F. Durham R.A. Schwarz R.D. Roher A.E. Walker L.C. Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice.J. Neurosci. 2000; 20: 3606-3611Crossref PubMed Google Scholar, Meyer-Luehmann et al., 2006Meyer-Luehmann M. Coomaraswamy J. Bolmont T. Kaeser S. Schaefer C. Kilger E. Neuenschwander A. Abramowski D. Frey P. Jaton A.L. et al.Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host.Science. 2006; 313: 1781-1784Crossref PubMed Scopus (716) Google Scholar). Jucker's finding raises an epistemologically significant question: if aggregation depends on the introduction of seeds and on the availability of the monomeric precursor, and if amyloid represents the primordial state of all proteins (Chiti and Dobson, 2006Chiti F. Dobson C.M. Protein misfolding, functional amyloid, and human disease.Annu. Rev. Biochem. 2006; 75: 333-366Crossref PubMed Scopus (4757) Google Scholar), wouldn't all proteins—under appropriate conditions—give rise to prionoids in the presence of sufficient precursor? The issues sketched above go well beyond AD and prions. There are many other diseases—not necessarily involving the nervous system—whose pathogenesis involves ordered aggregation of proteins, but for which there is no evidence of transmission between individuals. The best-studied of these are the systemic amyloidoses, which come about through the nucleation of some aggregation-prone proteins such as transthyretin and immunoglobulin light chains. Yet ordered protein aggregation is by no means confined to the “classical” amyloidoses and extends to a number of conditions, some of which have been rather unexpected. Type II diabetes is yet another disease whose pathogenesis may involve ordered protein aggregation. Evidence to support this idea was discovered over a century ago (Opie, 1901Opie E.L. The relation of diabetes mellitus to lesions of the pancreas: hyaline degeneration of the islets of Langerhans.J. Exp. Med. 1901; 5: 527-540Crossref PubMed Scopus (200) Google Scholar) but was largely forgotten until recently. It is now evident that aggregation of islet amyloid polypeptide (IAPP) is an exceedingly frequent feature of type II diabetes. IAPP amyloids damage the insulin-producing β cells within pancreatic islets and may crucially contribute to the pathogenesis of diabetes (Hull et al., 2004Hull R.L. Westermark G.T. Westermark P. Kahn S.E. Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes.J. Clin. Endocrinol. Metab. 2004; 89: 3629-3643Crossref PubMed Scopus (402) Google Scholar). It is unknown, however, whether IAPP deposition simply accrues linearly with IAPP production or whether it spreads prion-like from one pancreatic islet to the next. A body of recent work supports the idea that many aggregation proteinopathies are, in one way or another, transmissible. A recent report showed that α-synuclein is released from neurons and is then taken up by the neighboring cells, thereby aiding in a progressive spread of the protein (Desplats et al., 2009Desplats P. Lee H.J. Bae E.J. Patrick C. Rockenstein E. Crews L. Spencer B. Masliah E. Lee S.J. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein.Proc. Natl. Acad. Sci. USA. 2009; 106: 13010-13015Crossref PubMed Scopus (1011) Google Scholar, Lee et al., 2005Lee H.J. Patel S. Lee S.J. Intravesicular localization and exocytosis of alpha-synuclein and its aggregates.J. Neurosci. 2005; 25: 6016-6024Crossref PubMed Scopus (553) Google Scholar). When exogenously added to cultured cells, fluorescently labeled, recombinant α-synuclein was internalized from the extracellular milieu into the cytosol. Furthermore, injection of GFP-labeled mouse cortical neuronal stem cells into the hippocampus of α-synuclein-transgenic mice led to the efficient uptake of the host α-synuclein into the grafted cells after just 4 weeks. These findings are reminiscent of the observation that healthy fetal tissue, grafted into the brains of Parkinson's disease patients, acquired intracellular Lewy bodies. The latter phenomenon is somewhat anecdotal and has been disputed (Mendez et al., 2008Mendez I. Viñuela A. Astradsson A. Mukhida K. Hallett P. Robertson H. Tierney T. Holness R. Dagher A. Trojanowski J.Q. Isacson O. Dopamine neurons implanted into people with Parkinson's disease survive without pathology for 14 years.Nat. Med. 2008; 14: 507-509Crossref PubMed Scopus (333) Google Scholar), yet it would be entirely compatible with the hypothesis that α-synuclein aggregates are prionoids (Li et al., 2008Li J.Y. Englund E. Holton J.L. Soulet D. Hagell P. Lees A.J. Lashley T. Quinn N.P. Rehncrona S. Björklund A. et al.Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation.Nat. Med. 2008; 14: 501-503Crossref PubMed Scopus (1197) Google Scholar). A similar study conclusively demonstrated that exogenous α-synuclein fibrils induced the formation of Lewy body-like intracellular inclusions in vitro (Luk et al., 2009Luk K.C. Song C. O'Brien P. Stieber A. Branch J.R. Brunden K.R. Trojanowski J.Q. Lee V.M. Exogenous {alpha}-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.Proc. Natl. Acad. Sci. USA. 2009; 106: 20051-20056Crossref PubMed Scopus (545) Google Scholar). This study also showed that the conversion of the host cell α-synuclein was accompanied by dramatic changes, including hyperphosphorylation and ubiquitination of α-synuclein aggregates—thus recapitulating some key features of the human pathology. In experiments conceptually analogous to those discussed above, polyglutamine-containing protein aggregates similar to those present in Huntington's disease and in spinocerebellar ataxias exhibited prion-like propagation (Ren et al., 2009Ren P.H. Lauckner J.E. Kachirskaia I. Heuser J.E. Melki R. Kopito R.R. Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates.Nat. Cell Biol. 2009; 11: 219-225Crossref PubMed Scopus (326) Google Scholar). There, aggregation of huntingtin progressed from the extracellular space to the cytosol and eventually to the nucleus. What is more, similar phenomena occurred upon exposure of cells to Sup35 aggregates, which consist of a yeast protein for which there are no known mammalian paralogs. This suggests that the prionoid properties are intrinsic to amyloids and are not tied to the origin or function of their monomeric precursor protein. In another work, Tolnay and colleagues report a similar phenomenon in a mouse model of “tauopathy,” a neurodegenerative disease due to intraneuronal aggregation of the microtubule-associated tau protein (Clavaguera et al., 2009Clavaguera F. Bolmont T. Crowther R.A. Abramowski D. Frank S. Probst A. Fraser G. Stalder A.K. Beibel M. Staufenbiel M. et al.Transmission and spreading of tauopathy in transgenic mouse brain.Nat. Cell Biol. 2009; 11: 909-913Crossref PubMed Scopus (1119) Google Scholar). Aggregation-prone mutant tau, when extracted from the brain of transgenic mice, induced tauopathy in mice overexpressing wild-type tau. Assuming that tau pathology wasn't elicited by some indirect pathway (tau-overexpressing mice develop tangles when exposed to Aβ aggregates [Götz et al., 2001Götz J. Chen F. van Dorpe J. Nitsch R.M. Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Abeta 42 fibrils.Science. 2001; 293: 1491-1495Crossref PubMed Scopus (1188) Google Scholar]), these transgenic mice appear to behave like the Martian bottles, since tauopathy was not induced in mice expressing normal levels of tau. In yet another study, the microtubule binding part of the full-length tau was found to attack and penetrate cells when added exogenously, and this again induced host tau misfolding (Frost et al., 2009Frost B. Ollesch J. Wille H. Diamond M.I. Conformational diversity of wild-type Tau fibrils specified by templated conformation change.J. Biol. Chem. 2009; 284: 3546-3551Crossref PubMed Scopus (151) Google Scholar). This study also showed that aggregated intracellular Tau spontaneously transferred between two cocultured cell populations (Frost et al., 2009Frost B. Ollesch J. Wille H. Diamond M.I. Conformational diversity of wild-type Tau fibrils specified by templated conformation change.J. Biol. Chem. 2009; 284: 3546-3551Crossref PubMed Scopus (151) Google Scholar). In the case of both tau and polyglutamines, the protein aggregates appear to gain access to the cytosol and to cause further aggregation of their host counterparts—presumably by nucleation. The unifying characteristics of all these diseases is the aggregation of proteins into highly ordered stacks, termed amyloids irrespective of their size; the growth of these structures also exhibits generic features (Knowles et al., 2009Knowles T.P.J. Waudby C.A. Devlin G.L. Cohen S.I.A. Aguzzi A. Vendruscolo M. Terentjev E.M. Welland M.E. Dobson C.M. An analytical solution to the kinetics of breakable filament assembly.Science. 2009; 326: 1533-1537Crossref PubMed Scopus (737) Google Scholar) shared with a wide class of self-assembly phenomena characterized by elongation and fragmentation, such as the formation of analogous aggregates in micro-organisms and in vitro. Two conclusions can be drawn from the recent studies: (1) an unexpected number of amyloidogenic proteins can be released from affected cells in the form of extracellular amyloid seeds, and (2) even more surprisingly, these seeds can then re-enter other cells and nucleate the aggregation of their intracellular counterparts—in the cytosol or even in the nucleus. The biological and practical implications are far-reaching. On the one hand, cell therapies of aggregation diseases may be more difficult than anticipated, as the transplanted cells may undergo infection. A possible remedy could consist in the removal of the genes encoding the precursor of the offending proteins from the cells utilized for therapy—e.g., using the zinc-finger nuclease strategy (Hockemeyer et al., 2009Hockemeyer D. Soldner F. Beard C. Gao Q. Mitalipova M. DeKelver R.C. Katibah G.E. Amora R. Boydston E.A. Zeitler B. et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.Nat. Biotechnol. 2009; 27: 851-857Crossref PubMed Scopus (779) Google Scholar). On the other hand, a novel paradigm of amyloid pathogenesis is emerging from these data, whereby each prionoid behaves as a self-assembling and self-replicating nanomachine. Conversely, these findings raise a number of enigmas for which we are lacking any satisfactory answer. Whereas PrPC and the Aβ are luminally exposed, α-synuclein and tau are cytoplasmic—and huntingtin is even nuclear. Aggregates of both Aβ and PrPSc, as well as their monomeric precursors, are found in the extracellular space; it is hence intuitive that the nucleation process can propagate spatially across large distances. Instead, the propagation of cytoplasmic prionoids challenges our basic cell-biological understanding, since it posits that protein aggregates are released into the extracellular space and can subsequently reenter—and wreak havoc—in the cytosol of other cells. The release of cytosolic amyloids is supported by the amelioration of Lewy body pathology in α-synuclein transgenic mice immunized with human α-synuclein (Masliah et al., 2005Masliah E. Rockenstein E. Adame A. Alford M. Crews L. Hashimoto M. Seubert P. Lee M. Goldstein J. Chilcote T. et al.Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease.Neuron. 2005; 46: 857-868Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar). Similarly, anti-tau oligomer immunotherapy reduced brain pathology (Asuni et al., 2007Asuni A.A. Boutajangout A. Quartermain D. Sigurdsson E.M. Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements.J. Neurosci. 2007; 27: 9115-9129Crossref PubMed Scopus (380) Google Scholar), and immunization with mutant SOD1 led to clearance of SOD1 and delayed the onset of the disease in mice (Urushitani et al., 2007Urushitani M. Ezzi S.A. Julien J.P. Therapeutic effects of immunization with mutant superoxide dismutase in mice models of amyotrophic lateral sclerosis.Proc. Natl. Acad. Sci. USA. 2007; 104: 2495-2500Crossref PubMed Scopus (179) Google Scholar). All of these results indicate that cytosolic amyloids are somehow accessible to extracellular antibodies. This raises the question of how these proteins are released into the extracellular space (“cytosol to lumen”) and how they subsequently re-enter cellular cytosol (“lumen to cytosol”). Both events require trespassing lipid bilayer barriers—by no means a trivial feat for proteins, let alone high-molecular-weight aggregates. The release of cytosolic proteins into the extracellular milieu is by no means an exclusive feature of amyloids. While most secreted proteins follow the conventional ER-Golgi biosynthetic pathway, several proteins have been reported to be secreted through a noncanonical pathway (Muesch et al., 1990Muesch A. Hartmann E. Rohde K. Rubartelli A. Sitia R. Rapoport T.A. A novel pathway for secretory proteins?.Trends Biochem. Sci. 1990; 15: 86-88Abstract Full Text PDF PubMed Scopus (250) Google Scholar, Nickel, 2003Nickel W. The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.Eur. J. 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Reconstitution assays in inside-out vesicles (extracellular side inside and cytoplasm outside) indicate that cytosolic FGF-2 can directly translocate to the extracellular compartment in a temperature- and time-dependent manner. This translocation is also exhibited by galectin-1, but not by FGF-4 protein or MIFs, suggesting a certain degree of specificity. Polypeptides are transported across the endoplasmic reticulum bilayer by the Sec61 translocon and—in the opposite direction—by the retrotranslocon machinery (Lilley and Ploegh, 2004Lilley B.N. Ploegh H.L. A membrane protein required for dislocation of misfolded proteins from the ER.Nature. 2004; 429: 834-840Crossref PubMed Scopus (559) Google Scholar): one might therefore posit the existence of analogous transporters embedded in the plasma membrane. Could cytoplasmic prionoids utilize such hypothetical transporters? 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However, the precise mechanics of this type of secretion remains mysterious. Although the interaction of aggregates with lipids has been documented to occur in protein-free liposomes, its importance for translocation across biological membranes remains unclear. Apoptotic blebs are subcellular micelles that are released by dyi