17 August 2008. In mammals, molecular chaperones may help stave off neurodegenerative disease by steering proteins away from abnormal, toxic conformations. What mammals seem to be less adept at is reversing abnormal folding and protein aggregation once it has already occurred. Plants, fungi, and bacteria have a leg-up on the animal kingdom in this regard. They all possess orthologs of heat shock protein 104, a powerful, ATP-driven, protein remodeling engine. Hsp104 helps break up protein aggregates, returning individual components back to their normal form. Could a similar apparatus be engineered to resolve protein aggregates associated with neurodegenerative diseases such as Alzheimer and Parkinson diseases? In this week’s Journal of Clinical Investigation online, researchers report that Hsp104 can, in fact, break up amyloid and pre-amyloid fibrils of α-synuclein, the major constituent of the intracellular inclusions, or Lewy bodies, that characterize Parkinson disease. What’s more, introducing the chaperone into rats that generate copious α-synuclein inclusions protected dopaminergic cells that fall victim to synuclein toxicity. The findings, together with previous reports that Hsp104 can protect against other protein aggregates, hint that the chaperone could one day be exploited for therapeutic purposes.
Yeast Hsp104 was previously shown to reduce aggregation of mutant huntingtin (see Carmichael et al., 2000), and work from Susan Lindquist’s lab showed that it can also break up pre-amyloid prion oligomers (see Shorter and Lindquist, 2006). To test how Hsp104 might affect α-synuclein aggregation, Lindquist, at the Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, teamed up with Patrick Aebischer and colleagues at the Ecole Polytechnique Fédérale de Lausanne, in Switzerland. Lead author Christophe Lo Bianco and colleagues focused on a lentivirus rat model of PD in which overexpression of a mutant human α-synuclein gene (A30P) leads to selective loss of substantia nigra dopaminergic neurons. The researchers found that if Hsp104 is co-expressed with the human α-synuclein, it helps rescue the phenotype. Loss of tyrosine hydroxylase (TH)-positive dopaminergic cells six weeks after lentiviral treatment was significantly reduced (from about 30 to 13 percent cell loss). In addition, Hsp104 co-expression reduced loss of TH-positive nerve terminals projecting into the striatum by threefold (21 percent loss versus 7 percent in Hsp104-treated animals). The researchers also found that neurodegeneration in general, as judged by silver staining, was reduced by introduction of the chaperone.
Is the dopaminergic protection afforded by Hsp104 due to general chaperone activity or to a more specific reigning in of α-synuclein? To address this, Lo Bianco and colleagues examined rat brain specifically for α-synuclein pathology. Though it was difficult to distinguish between aggregates and general accumulation of the protein in this model, they did find that dense puncta of α-synuclein were much less evident in the animals expressing Hsp104 and that the number of cells harboring such entities fell from 55 to 31 percent. The researchers also found a 57 percent reduction in cells containing α-synuclein phosphorylated on serine 129. S129-phosphorylated synuclein is a major constituent of Lewy bodies.
The work indicates that Hsp104 can indeed protect against accumulation of α-synuclein aggregates, but does it simply prevent their formation, or can it reverse aggregates that have already formed? The authors used an in vitro approach to tackle this issue and the results suggest that the chaperone can do both. It retarded fibrillization of normal, wild-type α-synuclein and also of A30P, E46K, and A53T mutants, though it was not as effective in the latter case. It also prevented fibrillization of the phosphorylation incompetent S129A α-synuclein, and the S129-phosphorylation mimic S129E. To test if it can reverse, or remodel, already formed oligomers of α-synuclein, which, as is the case with amyloid-β, may be the most toxic form of the protein (see ARF related news story), the researchers incubated purified A30P α-synuclein oligomers with Hsp104. A30P oligomers are relatively stable, lasting 10 days or more in solution, but in the presence of the chaperone they were almost fully degraded within an hour. “Thus, Hsp104 eradicates α-syn A30P pre-amyloid oligomers, which are potentially the most toxic species that arise during A30P amyloidogenesis,” write the authors.
That yeast Hsp104 seems to work in vivo in mammals raises the possibility that it could be used as some kind of therapy for not only Parkinson disease, but many other diseases believed to result from formation of toxic amyloids, such as Alzheimer disease. The fact that Hsp104 works on α-synuclein, which bears little resemblance to the chaperone’s natural substrates such as yeast Sup35, Ure2, and Rnq1, suggests that “Hsp104 may specifically engage and remodel generic aspects of the cross-β amyloid form,” write the authors, though they note that further work is needed to determine if the chaperone reduces oligomers in vivo and to evaluate the safety of long-term expression of Hsp104 in neurons.
Lastly, there is the question of whether mammals have truly lost their version of the chaperone during evolution. As the authors write, though attempts to isolate such activity have not been successful, it is unclear whether mammalian cells express an analogous protein or not. Given that mammalian heat shock proteins Hsp70 and Hdj2 seem to augment the ability of Hsp104 to promote α-synuclein oligomer disassembly, a mammalian Hsp104-like molecule, should it exist, might offer a more tractable therapeutic target.—Tom Fagan.
Lo Bianco C, Shorter J, Régulier E, Lashuel H, Iwatsubo T, Lindquist S, Aebischer P. Hsp104 antagonizes alpha-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease. J. Clin. Invest. 2008, August 14 online. Abstract