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. 2011 Mar 1;108(9):3548-53.
doi: 10.1073/pnas.1017275108. Epub 2011 Feb 14.

Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells

Christian Münch  1 , John O'Brien, Anne Bertolotti
Affiliations

Prion-like propagation of mutant superoxide dismutase-1 misfolding in neuronal cells

Christian Münch et al. Proc Natl Acad Sci U S A. .

Abstract

Deposition of proteins of aberrant conformation is the hallmark of many neurodegenerative diseases. Misfolding of the normally globular mutant superoxide dismutase-1 (SOD1) is a central, early, but poorly understood event in the pathogenic cascade leading to familial forms of ALS. Here we report that aggregates composed of an ALS-causing SOD1 mutant penetrate inside cells by macropinocytosis and rapidly exit the macropinocytic compartment to nucleate aggregation of the cytosolic, otherwise soluble, mutant SOD1 protein. Once initiated, mutant SOD1 aggregation is self-perpetuating. Mutant SOD1 aggregates transfer from cell to cell with remarkable efficiency, a process that does not require contacts between cells but depends on the extracellular release of aggregates. This study reveals that SOD1 aggregates, propagate in a prion-like manner in neuronal cells and sheds light on the mechanisms underlying aggregate uptake and cell-to-cell transfer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mutant SOD1 aggregates penetrate inside neuronal cells. (A) Purified SOD1H46R (1.2 μg), unlabeled or labeled with Dylight dyes, analyzed by NuPAGE and stained with Coomassie brilliant blue. (B) Soluble (sol.) or aggregated (aggr.) SOD1H46R labeled with Dylight dyes, digested with 0.125% trypsin for 1 min where indicated, prior to native PAGE followed by immunoblot analysis with SOD1 polyclonal antibodies. (C) Neuro-2a take up labeled SOD1H46R aggregates in a time-dependent manner. Flow cytometry analysis of Neuro-2a cells inoculated with Dylight649-SOD1H46R aggregates (0.2 μM monomer equivalent); a.u., arbitrary units. (D) Confocal micrographs of cells untreated or inoculated with labeled SOD1H46R aggregates 15 min before fixing the cells. Nuclei were stained with H33258. Aggregates were estimated to be 2–4 μm inside the cell, not at the surface. Representative results of at least three independent experiments are shown. (E) Flow cytometry and confocal analysis of cells left untreated for 3 h after inoculation with Dylight649-labeled SOD1A4V, SOD1H46R, or SOD1L144F aggregates.
Fig. 2.
Fig. 2.
Mutant SOD1 aggregates are internalized by vesicles and escape the endocytic compartment to seed aggregation of the endogenous mutant protein. (A) SOD1H46R aggregates localize in membrane-enclosed vesicles. Confocal micrographs of cells after inoculation with Dylight488-SOD1H46R aggregates (green) for 5 min together with the fixable membrane dye FM 4-64FX (red). Nuclei were stained with H33258 dye. (B) Aggregates rapidly exit the FM 4-64FX marked vesicles. Same as in A except that cells were fixed 30 min after addition of aggregates and the FM 4-64FX dye. (C) Confocal micrographs of cells transiently expressing SOD1-GFP wild-type or SOD1H46R-GFP and inoculated with 0.2 μM (monomer equivalent) Dylight649-SOD1H46R aggregates, where indicated, for 15 h before fixing the cells. (D and E) Cells were exposed to Dylight649-SOD1H46R for 15 h, trypsinized, and cultured for 2 d before fixing, labeling with the polyubiquitin antibody FK2 where indicated, and confocal microscopy. Representative results of at least three independent experiments are shown.
Fig. 3.
Fig. 3.
Mutant SOD1 aggregates enter cells by macropinocytosis. (A) Confocal micrographs of Neuro-2a cells inoculated with 0.2 μM (monomer equivalent) Dylight649-SOD1H46R aggregates (red) and Alexa488-transferrin (green) for 5 min before fixing the cells. (B) The effect of inhibitors of endocytosis on SOD1H46R aggregate uptake. Cells either treated with the indicated drugs or expressing dominant-negative (DN) mutants of components of endocytic pathways, or lacking key components of endocytic pathways, were inoculated with 0.2 μM (monomer equivalent) Dylight649-SOD1H46R aggregates for 30 min. Aggregate uptake was monitored by flow cytometry. Data are means ± SD (n≥3) normalized to that of either untreated cells, mock transfected cells, or to the matching wild-type control cells when analyzing uptake in genetically modified cells. Conditions inhibiting macropinocytosis are highlighted in green. (C) Table recapitulating the effects of the different treatments used in B.
Fig. 4.
Fig. 4.
Induced mutant SOD1 aggregation is persistent and heritable. (A) Confocal micrographs of Neuro-2a cells stably expressing SOD1H46R-GFP left untreated or (B) transiently inoculated for 15 h with 0.2 μM (monomer equivalent) Dylight649-SOD1H46R aggregates (red). Neuro-2a cells were passaged 3 times per week and plated on coverslips 12 h before fixing at the indicated time after seeding. Nuclei were stained with H33258. Representative results of least three independent experiments are shown. (C) Cells (approximately 130 per condition) treated as in B were blindly scored for the presence of internalized or induced aggregates.
Fig. 5.
Fig. 5.
Transfer of aggregates from cell to cell is dependent on their release to the extracellular space. (A) Cells were inoculated separately with either Dylight488- or Dylight649-SOD1H46R aggregates, trypsinized to remove the inoculum, mixed, and cocultured for 15 h before flow cytometry analysis. (B) As in A, except that cells were left for 6 h after digestion of the inoculum with trypsin before they were cocultured. (C) Confocal micrographs of cells treated as in A. (D) Cells inoculated separately with either Dylight488 or Dylight649-SOD1H46R aggregates were cocultured in a 24 multiwell dish for 15 h, with increasing volumes of medium and analyzed by flow cytometry. Transfer was scored as the number of Dylight488 and Dylight649 double-positive cells. (E) Flow cytometry analysis of Neuro-2a cells containing Dylight488-SOD1H46R aggregates inoculated with conditioned media from Neuro-2a cells with internalized Dylight648-SOD1H46R aggregates. (F) Same as in A except that the two cell populations were cocultured in a chamber separated by a 0.4-μm filter; a.u., arbitrary units. Representative results of at least three independent experiments are shown.
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