Keystone: Tau, Huntingtin—Do Prion-like Properties Play a Role in Disease?
Could prion-like properties of the microtubule binding protein tau explain the phenotypic diversity and the characteristic progression of different tauopathies, such as Alzheimer disease (AD) and frontotemporal dementia? That provocative idea was raised by Marc Diamond, University of California, San Francisco, at the Keystone symposium, “Neurodegeneration: New Molecular Mechanisms,” held 17-22 February in Keystone, Colorado. He was not alone in addressing the possibility. Fellow Californian Ron Kopito from Stanford University suggested prion-like properties might help explain the toxicity of proteins with polyglutamine expansions, such as huntingtin.
The prion-like property Diamond and Kopito referred to is the ability of misfolded proteins to seed aggregation, a property shared by amyloid-β and other amyloidogenic proteins. This is typically studied in a cell-autonomous fashion, noted Diamond. But what if the seeds could get from one cell to another? Could they then promote aggregation of protein in otherwise healthy cells, a la prions, and could that explain distinct patterns of progression seen in diverse diseases?
To explore this idea, Diamond and colleagues focused on tau. Fibrillar, toxic tau is typically found inside glia and neurons, as in the neurofibrillary tangles of AD, but extracellular NFTs (sometimes called “ghost” tangles) have been found as well, suggesting that the protein has the means of surviving outside the cell and possibly “infecting” others nearby. (Phosphorylated tau is also found in the cerebrospinal fluid, which suggests the protein is not restricted to intracellular space). Tau is also associated with 20 or more different tauopathies, each with their own characteristic pathology and progression, and different disease-associated tau mutations can form distinct protein conformations (see Von Bergen et al., 2000), raising the possibility that like prions, there are different strains, or conformers, of tau with distinct disease-causing characteristics.
To test whether tau has prion-like properties, Diamond and colleagues studied tau fibrils formed from wild-type and mutant (P301L/V337M) protein. He showed that when either aggregates in vitro, it forms fibrils with distinct conformations. “This is not surprising, since the amino acid sequences are different,” said Diamond. “But can you drive the wild-type into a different conformation by seeding with the mutant?” The answer appears to be yes. He showed that wild-type protein forms what he called WT* when seeded with mutant fibrils. WT* has a distinct secondary structure, as seen by Fourier transform infrared and circular dichroism spectroscopies. It also exhibits different fragility under the atomic force microscope than do wild-type fibrils.
The work, some of which was recently published in the February 6 Journal of Biological Chemistry online (see Frost et al., 2009), indicates that tau can exist in at least two conformations. Prions exist in different conformations that are linked to distinct infectious forms (see Tanaka et al., 2004); Diamond’s work suggests the same may be true for tau. These findings “provide a plausible mechanism for phenotypic diversity seen in tauopathies,” he said, though he hastened to add that the existence of different conformers in patient material needs to be confirmed.
If tau can exist in different conformations, could those conformations be transmitted from cell to cell? Diamond and colleagues tested this using rhodamine dye-tagged tau. He showed that tau does indeed gain entry into various cell types, including primary neuronal cultures, and that it may be taken up in an active manner, since the protein often colocalizes with endocytic markers. But are specific misfolding patterns propagated? This was tested by treating cells expressing yellow fluorescent protein (YFP)-tagged normal tau with fibrils made from a fragment of tau comprising the microtubule binding region (MTBR) and a hemagglutinin (HA) tag. The MTBR fibrils caused aggregation of normal tau, as seen by the appearance of yellow puncta in the cells, and these colocalized with the HA-MTBR fibrils taken up by the cells. To test if this conversion can be propagated from cell to cell, Diamond mixed cells expressing either YFP-labeled tau or red fluorescent protein (RFP)-labeled tau. He showed that after co-culturing the cells and then separating them, those that only express RFP-labeled tau nonetheless have green puncta—indicative of aggregation—suggesting that YFP-tau has made it across the cellular divide. The experiment suggests that tau can move from one cell to another, raising the possibility that in some cases an abnormal form might move in and potentially alter aggregation of normal tau.
Diamond concluded his talk by reiterating the idea that pathogenic tau, such as hyperphosphorylated or mutated tau, is conformationally diverse, aggregation-prone, can corrupt normal tau, and can spread between cells. He suggested that if this mechanism is common for other aggregating proteins, it might explain local propagation of cellular dysfunction between neurons and glia—as in ALS—or network degeneration, where protein from one cell can cross a synapse, for example, and corrupt normal protein in a postsynaptic cell. He also suggested that what makes prions unique is not their ability to propagate, but their inherent stability, which makes them much more likely to be infectious agents.
Kopito also drew comparisons between prions and another amyloidogenic protein, polyglutamate-expanded huntingtin. Prions self-assemble, have sequence and strain specificity, and can propagate from cell to cell. Expanded huntingtin shares the first two properties, said Kopito, but it was not clear if it can propagate from cell to cell, he said. To test this, Kopito, together with postdoc Pei-Hsien Ren and colleagues in his lab, used a nucleation-based assay using synthetic polyglutamine peptides (KKQnKK). In this assay, cells expressing a cyan fluorescent protein (CFP)-tagged HttQ25 peptide (which does not aggregate) were treated with fibrils of a rhodamine-tagged Q44 peptide. The idea was that if Q44 fibrils do not behave as prions, then they would probably be taken up by the cell into lysosomes, where they would be degraded, and not interact with normal cytosolic huntingtin. However, if they could gain access to the cytosol, then they could co-opt the Q25 proteins into their fibrils, effectively spreading protein misfolding. In fact, this is what Kopito and colleagues saw. Within one hour of treating HEK293 cells, the normally diffuse CFP-tagged Htt fragment redistributed into puncta that colocalized precisely with Q44 aggregates taken up from outside. After 24 hours, nearly 100 percent of the cells exhibited this phenotype. The effect is sequence specific, since neither aggregates of Aβ nor Sup35, the yeast prion, could convert CFP-HttQ25 to a fibrillar form.
So do polyglutamine expanded proteins behave like prions? To test this, Kopito and colleagues looked at another prion property—inheritance. Different prion strains, such as found in the yeast prion Sup35 (see ARF related news story), confer different phenotypes on their host, and these phenotypes can be propagated from generation to generation. Ren and colleagues tested if that might be true in cells infected with polyglutamine expanded huntingtin. They treated CFP-HttQ25-expressing HEK293 cells with KKQ44KK aggregates and then allowed the cells to divide. While initially all the cells had CFP puncta, indicative of induced aggregation of the httQ25 chimera, the number of cells with puncta declined with each round of cell division, consistent with dilution of the original innoculum. But curiously, after about 30 generations, the number of CFP-Htt puncta per cell rose and was significantly higher than if the cells were infected with a control treatment, such as Aβ aggregates. The experiment suggested that the aggregation phenotype can be passed on to the next generation. Persistent aggregation is a new and inheritable phenotype, suggested Kopito.
So are tau, mutant huntingtin, and perhaps other amyloidogenic proteins really prions? Kopito was hesitant to call polyglutamine expanded proteins prions, preferring the term prion-like. Heather True, Washington University School of Medicine, St. Louis, Missouri, who studies yeast prions and was not involved in either Kopito’s or Diamond’s work, was at the Keystone symposium. In a post-meeting interview with ARF, True said, “We can think about these proteins as having prion-like mechanisms, but it is hard to use the word ‘prion’ per se.” One of the main differences is localization, she said. “It is an accessibility issue more than anything else. I think that the underlying mechanisms are really very similar, but realistically, with PrP being a cell surface protein, the accessibility and availability of substrate, which really makes for a strong persistent infection, is a huge factor.”—Tom Fagan.
- von Bergen M, Friedhoff P, Biernat J, Heberle J, Mandelkow EM, Mandelkow E. Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5129-34. PubMed.
- Frost B, Ollesch J, Wille H, Diamond MI. Conformational diversity of wild-type Tau fibrils specified by templated conformation change. J Biol Chem. 2009 Feb 6;284(6):3546-51. PubMed.
- Tanaka M, Chien P, Naber N, Cooke R, Weissman JS. Conformational variations in an infectious protein determine prion strain differences. Nature. 2004 Mar 18;428(6980):323-8. PubMed.
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Florida Institute of Technology
For those who think of amyloid fiber formation as a prion-like process, you may want to review a paper I published (Xu, 2007). You can send your comments directly to me, e-mail email@example.com, or comment on this forum. It seems to me that we are ignoring the work of a much larger group of scientists, chemists, and physicists.
I copy here the abstract: “Protein amyloid fibers are often found to have a β-pleated sheet structure regardless of their sequence, leading some to believe that it is the molecule’s misfolding that leads to aggregation. In this article, an alternative model is introduced for the amyloid community to consider, that fiber formation is a surface-energy minimization process, starting with the generation of colloidal particles and their linear assembly, and ending with structural evolution of the aggregates into mature fibers. We propose that aggregation drives conformational change and that a conformational change is not essential to initiate the aggregation process.”
Xu S. Aggregation drives "misfolding" in protein amyloid fiber formation. Amyloid. 2007 Jun;14(2):119-31. PubMed.
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