Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms

Abstract

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

Keywords: disease-specific epitope; intercellular transmission; prion-like; protein misfolding.

Fig. 1.

Aggregated SOD1 is released from dead or dying cells and/or actively released as part of exosomes. (A) Cell death of GFP-positive cells after 72 h posttransfection was examined by flow cytometry. Cells stained with PI were scored as dead and these cells expressed as a proportion of total GFP-positive cells. The results are means ± SE of three independent experiments. *P < 0.05 compared with medium from nontransfected (NT) cells, as determined by unpaired t test. (B) Uptake of pelletable SOD1–GFP by naïve NSC-34 cells following incubation with conditioned media was measured by flow cytometry. The data shown is calculated from triplicate experiments. Error bars represent SEM. *P < 0.05 compared with medium from nontransfected (NT) cells, as determined by unpaired t test. (C) Detection of oligomeric SOD1 released into media 72 h posttransfection. The white arrowhead indicates SOD1–GFP monomer whereas the black arrowhead indicates SOD1–GFP dimer. The area indicated by an asterisk represents SDS-resistant oligomeric SOD1 species. Immunoblot probed with GFP pAb. (D) Filter-trap assay and anti-GFP immunoblot to detect SOD1–GFP aggregates in conditioned media. The filter trap was performed in triplicate and is quantified in E. Values obtained from the filter trap assay are the mean average of three independent experiments. Error bars represent SEM. *P < 0.05 compared with negative controls (NT and EGFP). (F) Quantitative analysis of aggregated human SOD1 internalization into NSC-34 cells using flow cytometry. Cells were either incubated with PBS (gray) or aggregated HuWtSOD1 (red line). (G) Visualization of exosomes secreted by NSC-34 cells by negative staining electron microscopy. (Scale bar: 100 nm.) (H) Immunogold labeling using 3H1 antibody shows surface-localized misfolded SOD1 on exosomes secreted by stably transfected NSC-34 cells. (Scale bar: 50 nm.) (I) Quantification of exosomal and proximal free misfolded SOD1 secreted by NSC-34 cells. (J) NSC-34 cells were treated with NSC-34–derived exosomes for 30 min and fixed. Quantitative analysis of SOD1–GFP internalization into NSC-34 cells using flow cytometry. Cells were either incubated with PBS (gray) or HuWtSOD1–GFP exosomes (red line).

Fig. 2.

Mutant SOD1-mediated HuWtSOD1 misfolding is transmissible between cells. (A) IP of lysates from naïve HEK cells cultured in the presence of conditioned medium from one, three, and five passages of transduction assays initiated by different SOD1 variants. mIgG2a, mouse IgG2a isotype control; rIgG, rabbit mixed IgG control; SOD1, pan-SOD1 antibody; 3H1 and 10C12, misfolded SOD1-specific antibodies. (B) SOD1 knockdown abolishes detectable misfolding. IP of lysates from HEK cells with knocked down SOD1 expression cultured in the presence of conditioned medium from HEK cells transfected with empty vector control (EV), SOD1-G85R, SOD1-G127X, or HuWtSOD1. (C) IP of lysates from naïve primary neural cell cultures derived from embryonic spinal cord of HuWtSOD1 transgenic or nontransgenic littermate (n-Tg) mice incubated in the presence of conditioned from SOD1-G127X or empty vector (EV)-transfected HEK293 cells.

Fig. 3.

Misfolded SOD1 is detectable in the culture media of cells transfected with SOD1 constructs. (A) Misfolded SOD1 was detected by IP in both the pellet (P) and supernatant (S) fractions of centrifugated conditioned media, but majority was detected in the P fraction. C-less GX, cysteine-less version of G127X SOD1. This was confirmed by Biacore detection of misfolded SOD1 in the P fraction by 3H1 capture (B). Values represent the average of seven independent experiments. An asterisk indicates significant values. Statistical significance determined by unpaired t test, compared with EV control (significant P values shown). Error bars represent SD.

Fig. 4.

SOD1 antibodies can block misfolding transmission. IP of lysates from naïve HEK cells cultured in the presence of conditioned medium from G127X-transfected HEK cells (A), G85R-transfected HEK cells (B), or HuWtSOD1-transfected HEK cells (C) immunologically pretreated with either rabbit mixed IgG (rIgG), pan-SOD1 pAb (SOD1), or DSE mAbs 3H1 or 10C12, all at 20 μg/mL. Corresponding quantitation is shown to the right of each immunoblot. Values represent the mean average of four independent experiments. Error bars represent SD. An asterisk indicates statistically significant blocking of wtSOD1 misfolding transmission by specific antibodies as determined by ANOVA analysis followed by Tukey (honest significant difference) multiple comparison. For G127X experiment, P = 0.0048; for G85R experiment, P = 0.0100; for HuWtSOD1 experiment, P = 0.032.

Fig. 5.

Misfolded SOD1 is detectable in the spinal cords of both FALS and SALS patients. IHC using antibodies against misfolded SOD1 in cases of FALS with SOD1 mutations (A–D) and SALS (E and F). In cases with SOD1 mutations, neuronal cytoplasmic inclusions and neurites (arrow) were labeled in the primary motor cortex (A) and ventral gray matter of the spinal cord (B and C). The spinal gray matter (D) also contained numerous swollen axons. Immunopositive axons of normal caliber with a visible myelin sheath were present in the corticospinal tracts (arrows in E) and motor nerve roots (arrows in F) in sporadic cases. IHC was performed using 10E11C11 (A, B, and D–F) and 3H1 (C) antibodies. (Scale bars: 20 µm in A and D, 30 µm in B and C, and 12 µm in E and F.) (G) IP of 10% (wt/vol) spinal cord homogenate in PBS using DSE mAbs, normalized to equal concentrations of SOD1. Samples were preincubated at 37 °C for 30 min in the presence or absence of 200 µg/mL PK. All immunoblots were probed with pan-SOD1 pAb. C, cervical spinal cord; T, thoracic spinal cord.