Soluble polyglutamine oligomers formed prior to inclusion body formation are cytotoxic.
Expanded polyglutamine (polyQ) repeats cause neurodegenerative disorders, but their cytotoxic structures remain to be elucidated. Although soluble polyQ oligomers have been proposed as a cytotoxic structure, the cytotoxicity of soluble polyQ oligomers, not inclusion bodies (IBs), has not been proven in living cells. To clarify the cytotoxicity of soluble polyQ oligomers, we carried our fluorescence resonance energy transfer (FRET) confocal microscopy and distinguished oligomers from monomers and IBs in a single living cell. FRET signals were detected when donor and acceptor fluorescent proteins were attached to the same side, not the opposite side, of polyQ repeats, which agrees with a parallel beta-sheet or a head-to-tail cylindrical beta-sheet model. These FRET signals disappeared in semi-intact cells, indicating that these polyQ oligomers are soluble. PolyQ monomers assembled into soluble oligomers in a length-dependent manner, which was followed by the formation of IBs. Notably, survival assay of neuronally differentiated cells revealed that cells with soluble oligomers died faster than those with IBs or monomers. These results indicate that a length-dependent formation of oligomers is an essential mechanism underlying neurodegeneration in polyQ-mediated disorders.
Takahashi
Toshiaki
T
School of Health Sciences, Faculty of Medicine, Niigata University, 1-757 Asahimachi, Niigata 951-8122, Japan.
Kikuchi
Shinya
S
Katada
Shinichi
S
Nagai
Yoshitaka
Y
Nishizawa
Masatoyo
M
Onodera
Osamu
O
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000071058:Huntingtin Protein; D002479:Inclusion Bodies / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D017931:DNA Primers; C086055:HTT protein, human; D000071058:Huntingtin Protein; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C095278:atrophin-1; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C097188:polyglutamine