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Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves.

ABSTRACT: Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this purpose, provided their specificity and selectivity are well-characterized. Here, we characterized three recently introduced antimisfolded SOD1 antibodies and compared them with two commercial, antimisfolded SOD1 antibodies raised against the fALS-linked variant G93A-SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti-SOD1 antibodies expected to be insensitive to misfolding. We asked to what extent the antibodies could distinguish between WT and variant SOD1 and between native and misfolded conformations. WT, G93A-SOD1, or E100K-SOD1 were incubated under aggregation-promoting conditions and monitored using thioflavin-T fluorescence, electron microscopy, and dot blots. WT and G93A-SOD1 also were analyzed using native-PAGE/Western blot. The new antimisfolded SOD1 and the commercial antibody B8H10 showed variable reactivity using dot blots but generally showed maximum reactivity at the time misfolded SOD1 oligomers were expected to be most abundant. In contrast, only B8H10 and the control antibodies were reactive in Western blots. Unexpectedly, the polyclonal antibodies showed strong preference for the misfolded form of G93A-SOD1 in dot blots. Surprisingly, antimisfolded SOD1 antibody C4F6 was specific for the apo form of G93A-SOD1 but insensitive to misfolding. Antibody 10C12 showed preference for early misfolded structures, whereas 3H1 bound preferentially to late structures. These new antibodies allow distinction between putative early- and late-forming prefibrillar SOD1 oligomers.

SUBMITTER: Atlasi RS

PROVIDER: S-EPMC6475120 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves.

Atlasi Ryan S RS Malik Ravinder R Corrales Christian I CI Tzeplaeff Laura L Whitelegge Julian P JP Cashman Neil R NR Bitan Gal G

ACS chemical biology 20180830 9

Mutations in Cu/Zn-superoxide dismutase (SOD1) gene are linked to 10-20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations cause misfolding and self-assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. The molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization of misfolded SOD1 is particularly challenging because of its metastable nature. Antibodies against misfolded SOD1 are useful tools for this p ...[more]

PMID: 30110532

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Project description:BACKGROUND:Prior studies in C. elegans demonstrated that the expression of aggregation-prone polyglutamine proteins in muscle wall cells compromised the folding of co-expressed temperature-sensitive proteins, prompting interest in whether the accumulation of a misfolded protein in pathologic features of human neurodegenerative disease burdens cellular proteostatic machinery in a manner that impairs the folding of other cellular proteins. METHODS:Mice expressing high levels of mutant forms of tau and ?-synuclein (?Syn), which develop inclusion pathologies of the mutant protein in brain and spinal cord, were crossed to mice expressing low levels of mutant superoxide dismutase 1 fused to yellow fluorescent protein (G85R-SOD1:YFP) for aging and neuropathological evaluation. RESULTS:Mice expressing low levels of G85R-SOD1:YFP, alone, lived normal lifespans and were free of evidence of inclusion pathology, setting the stage to use this protein as a reporter of proteostatic function. We observed robust induction of G85R-SOD1:YFP inclusion pathology in the neuropil of spinal cord and brainstem of bigenic mice that co-express high levels of mutant tau in the spinal axis and develop robust spinal tau pathology (JNPL3 mice). In contrast, in crosses of the G85R-SOD1:YFP mice with mice that model spinal ?-synucleinopathy (the M83 model of ?Syn pathology), we observed no G85R-SOD1:YFP inclusion formation. Similarly, in crosses of the G85R-SOD1:YFP mice to mice that model cortical tau pathology (rTg4510 mice), we did not observe induction of G85R-SOD1:YFP inclusions. CONCLUSION:Despite robust burdens of neurodegenerative pathology in M83 and rTg4510 mice, the introduction of the G85R-SOD1:YFP protein was induced to aggregate only in the context of spinal tau pathology present in the JNPL3 model. These findings suggest unexpected specificity, mediated by both the primary protein pathology and cellular context, in the induced "secondary aggregation" of a mutant form of SOD1 that could be viewed as a reporter of proteostatic function.

Project description:BackgroundCopper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2).MethodsThe mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay.ResultsWe observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death.ConclusionsCuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.

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Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves.

Publications

Investigation of Anti-SOD1 Antibodies Yields New Structural Insight into SOD1 Misfolding and Surprising Behavior of the Antibodies Themselves.

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