Spinal cord homogenates from SOD1 familial amyotrophic lateral sclerosis induce SOD1 aggregation in living cells

Abstract

Mutant Cu/Zn superoxide dismutase (SOD1) can confer its misfolding on wild-type SOD1 in living cells; the propagation of misfolding can also be transmitted between cells in vitro. Recent studies identified fluorescently-tagged SOD1G85R as a promiscuous substrate that is highly prone to aggregate by a variety of templates, in vitro and in vivo. Here, we utilized several SOD1-GFP reporter proteins with G37R, G85R, or G93A mutations in SOD1. We observed that human spinal cord homogenates prepared from SOD1 familial ALS (FALS) can induce significantly more intracellular reporter protein aggregation than spinal cord homogenates from sporadic ALS, Alzheimer’s disease, multiple system atrophy or healthy control individuals. We also determined that the induction of reporter protein aggregation by SOD1-FALS tissue homogenates can be attenuated by incubating the cells with the SOD1 misfolding-specific antibody 3H1, or the small molecule 5-fluorouridine. Our study further implicates SOD1 as the seeding particle responsible for the spread of SOD1-FALS neurodegeneration from its initial onset site(s), and demonstrates two potential therapeutic strategies for SOD1-mediated disease. This work also comprises a medium-throughput cell-based platform of screening potential therapeutics to attenuate propagated aggregation of SOD1.

Tryptophan residues in TDP-43 and SOD1 mediate the cross-seeding and toxicity of SOD1

“…We also show that induced conversion of SOD1 species by TDP-43, in both HEK293FT cells and zebrafish, is specified by specific tryptophan residues in both proteins, militating for a direct proteinprotein interaction. For SOD1, the aggregation of SOD1 G85R -GFP (in cultured cells) and cross-seeding of wild-type SOD1 (in zebrafish) is dependent on surface-exposed SOD1 Trp32, similar to earlier studies of SOD1-SOD1 propagated misfolding (10,11,13,14,20). Cross-seeding of wild-type SOD1 by TDP-43 ∆ NLS and wild-type TDP-43, and SOD1 G85R -GFP aggregation, are all dependent on the natively solventexposed RRM1 Trp172, and the misfolding/aggregation-induced exposure of Trp68 by loss-of-structure of the TDP-43 N-terminal domain.…”

“…Since SOD1 seeding and misfolding propagation is dependent on its single tryptophan residue, Trp32 (10,13,14), we speculated that TDP-43 might induce SOD1 misfolding and aggregation via one or more of its six Trp residues ( Fig 1A). Co-transfection of HEK293FT cells with SOD1 G85R -GFP reporter (13,20) and wild-type TDP-43 or nuclear localization signal mutant TDP-43 ∆ NLS revealed that SOD1 G85R -GFP aggregation was again strongly induced by these cytosolically mislocalizing and aggregating TDP-43 variants ( Fig 1B), supported by live-cell kinetic analysis ( Fig 1C). The induction of SOD1 G85R -GFP aggregation by wild-type TDP-43 or TDP-43 ∆NLS was confirmed independently by flow cytometry analysis showing a significant 3-fold increase in the induction of SOD1 G85R -GFP aggregation (Fig 1D and F).…”

“…The abundance of cells with induced aggregation of SOD1 G85R -GFP reporter protein was determined using flow cytometry (13,20). In order to identify only those cells that not only express SOD1 G85R -GFP, but where this report protein is indeed aggregated, we permeabilized the cells using 0.03% saponin in ice-cold PBS for 10 minutes to allow soluble SOD1 G85R -GFP to leak out of cells (13,20). Cells were then washed once using cold PBS and were stored on ice until analysis on LSRII (BD Biosciences, USA).…”

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

“…Further studies of the mechanism by which Trp 32 in SOD1 mediates the assembly of mutant SOD1 into larger aggregates may reveal potential avenues for therapy. Indeed, a recent study demonstrated that 5-fluoruridine, a compound that binds to SOD1 in a pocket that includes Trp 32, can attenuate the induced aggregation of SOD1:GFP in cultured cell models [73]. It may be of interest to generate transgenic mice that express mutant SOD1 with Trp 32 mutations to further explore the role of this residue in the macromolecular interactions that produce mutant SOD1 aggregates.…”

“…The mutation of Trp 32 to Ser potently inhibited the ability of misfolded WT SOD1 to propagate between cells in culture [57,68,69]. Small molecules that bind near Trp 32 can block the seeding of mutant SOD1 aggregation in cell culture models [68,69,73]. In the present study, we used a combination of cell culture models and in vivo seeding models to examine the role of Trp 32 in modulating mutant SOD1 aggregation.…”