In vitro recapitulation of aberrant protein inclusions in neurodegenerative diseases

Abstract

Aberrant protein aggregates in affected brain cells of patients with neurodegenerative diseases are a well-known hallmark, but although the formation of these inclusions is an important pathogenic event, the mechanism involved remains unclear. We have recently established a simple method to introduce protein fibrils into cultured cells as seeds for protein aggregation, and we showed that intracellular soluble α-synuclein or tau can aggregate in cultured cells dependently upon seeds introduced in this way. Seeded aggregation of α-synuclein induced necrotic cell death, which was suppressed by the addition of various polyphenols. Our cellular models are expected to be valuable tools not only for elucidating the molecular mechanisms of onset of neurodegenerative diseases, but also for drug discovery. 

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In patients with neurodegenerative disorders, intracellular aberrant protein inclusions are often found in the brain, including neurofibrillary tangles in Alzheimer's disease or Lewy bodies in Parkinson's disease and dementia with Lewy bodies. These aberrant protein aggregates are often observed in the most affected regions of diseased brains, suggesting they may cause neuronal cell death, leading to onset of these diseases. Tau and α-synuclein are well-known cytosolic proteins that are the main components of neurofibrillary tangles and Lewy bodies, respectively. They are soluble and natively unfolded proteins, and it remains unclear how they become aggregated in neuronal cells. Indeed, intracellular aggregate formation of these proteins does not occur when cultured cells are transfected with expression plasmids encoding these proteins. On the other hand, many in vitro studies using recombinant proteins, such as Abeta, tau, α-synuclein or poly glutamine-containing protein, have shown that these proteins are readily aggregated into fibrils in the presence of seeds for aggregation. These findings prompted us to examine whether seeds-dependent aggregation would occur in cultured cells. Thus, we aimed to introduce protein fibrils into cultured cells as seeds for aggregation.

Transfection of plasmid DNA into cultured cells is conducted routinely by the use of liposomes of polycationic and neutral lipids in water, based on the principle of cell fusion. Several commercially available reagents such as Lipofectoamine, Lipofectamine 2000 or FuGENE6 are available to efficiently transfect plasmid DNA into cultured cells. We tested whether these transfection reagents could transfect cultured SH-SY5Y cells not only with plasmid DNA, but also with protein fibrils. After much trial and error, we finally succeeded in transfecting α-synuclein fibrils into these cells using Lipofectamine reagent. We found that the introduced recombinant α-synuclein fibrils are phosphorylated at Ser129 in cultured cells, indicating that they had been introduced by Lipofectamine.1 Interestingly, monomeric and oligomeric α-synuclein could not be introduced by the use of Lipofectamine. We applied for a patent covering the use of Lipofectamine for transduction of recombinant protein fibrils into cultured cells in 2005 (patents pending in the United States: 12/086124, the European Union: 06834541.2 and Japan: 2007-549210). Recently, other groups have also reported introduction of fibrillar protein into cultured cells with or without specific reagents.2–5

Next, we examined whether intracellular α-synuclein can be aggregated dependently upon introduced seeds. When α-synuclein fibrils mixed with Lipofectamine were introduced into cells transiently expressing α-synuclein, phosphorylated and ubiquitinated α-synuclein inclusions (∼10 µm in diameter) were observed by means of confocal laser microscopy, indicating that plasmid-derived soluble α-synuclein formed aggregates in the presence of exogenous α-synuclein fibrils in cells, and these inclusions resembled Lewy bodies in diseased brains. Others have also reported that α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells.4 On the other hand, introduced tau fibrils were also shown to act as seeds for intracellular aggregation of plasmid-derived soluble tau protein. Interestingly, we found that fibrils composed of 3-repeat tau isoform serve as seeds for intracellular aggregation of soluble 3-repeat tau, but not soluble 4-repeat tau and fibrils of 4-repeat tau seed serve as seeds for aggregation of soluble 4-repeat tau, but not soluble 3-repeat tau. Likewise, introduction of α-synuclein fibrils did not elicit intracellular tau aggregation in cells and soluble α-synuclein did not form intracellular aggregates in the presence of any tau fibrils. These results clearly indicate that intracellular protein aggregation is highly dependent on the species of protein fibril seeds. Now, we are examining whether detergent-insoluble fractions prepared from several diseased brains can be introduced into cells by Lipofectamine and can serve as seeds for intracellular aggregate formation of soluble α-synuclein, tau or TDP-43.

Does the formation of these inclusions lead to cell death or toxicity? The answer is yes. We observed non-apoptotic cell death in cells harboring these inclusions. In these cells, proteasome activity was found to be significantly reduced. This suppression may be related to the cause of cell death. Furthermore, we showed that cell death in cells with α-synuclein inclusions is effectively suppressed by the addition of various small molecules to the culture medium; polyphenols such as exifone and gossypetin were the most effective, suggesting that these compounds may be possible new drugs for the treatment of neurodegenerative diseases.

Our study strongly supports a seed-dependent mechanism for the formation of the intracellular protein aggregates. Recently, the intercellular transfer of inclusions made of tau,3,6 α-synuclein2,7,8 and huntingtin9 has been reported, suggesting the existence of mechanisms reminiscent of those by which prions spread through the nervous system. It remains to be clarified whether the incorporation of amyloid seeds into neurons or glial cells, as shown in our study, also occurs in vivo, but our results strongly suggest that extracellular aggregates may be taken up into neurons by endocytosis or under certain specific conditions. Therefore, it may be crucial to inhibit not only the production of intracellular amyloid seeds, but also their spread into extracellular space and their propagation. Vaccination against α-synuclein10 or tau may be an effective treatment, together with the inhibition of intracellular aggregates formation with small-molecular compounds, for the therapy of neurodegenerative diseases.

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