Small peptides against the mutant SOD1/Bcl-2 toxic mitochondrial complex restore mitochondrial function and cell viability in mutant SOD1-mediated ALS.

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in 20% of familial cases (fALS). Mitochondria are one of the targets of mutant SOD1 (mutSOD1) toxicity. We previously demonstrated that at the mitochondria, mutSOD1 forms a toxic complex with Bcl-2, which is then converted into a toxic protein via a structural rearrangement that exposes its toxic BH3 domain (Pedrini et al., 2010). Here we now show that formation of this toxic complex with Bcl-2 is the primary event in mutSOD1-induced mitochondrial dysfunction, inhibiting mitochondrial permeability to ADP and inducing mitochondrial hyperpolarization. In mutSOD1-G93A cells and mice, the newly exposed BH3 domain in Bcl-2 alters the normal interaction between Bcl-2 and VDAC1 thus reducing permeability of the outer mitochondrial membrane. In motor neuronal cells, the mutSOD1/Bcl-2 complex causes mitochondrial hyperpolarization leading to cell loss. Small SOD1-like therapeutic peptides that specifically block formation of the mutSOD1/Bcl-2 complex, recover both aspects of mitochondrial dysfunction: they prevent mitochondrial hyperpolarization and cell loss as well as restore ADP permeability in mitochondria of symptomatic mutSOD1-G93A mice.

In vivo and in vitro determination of cell death markers in neurons.

Mitochondria are key regulators of cellular death. The mitochondrial membranes contain essential enzyme complexes for maintaining metabolic homeostasis and meeting the energy requirements of the cell (Tait and Green, Nat Rev Mol Cell Biol 11:621-632, 2010 and Galluzzi et al., Apoptosis 12:803-813, 2007). Thus, any perturbation of outer or inner mitochondrial membranes can lead to disruptions in the normal fluxes of key ions and metabolic proteins (i.e., ADP/ATP exchange), leading to eventual cellular death. In addition to maintaining cellular viability, mitochondria play a critical role in the initiation of programmed cell death. As initiators of the cell death process, key mitochondrial proteins [Cytochrome C (Cyt C) one of the most well-studied among them] are released from the intermembrane space during early cell death events eventually leading to caspase activation. Release of Cyt C is a crucial step during cellular death (Tait and Green, Nat Rev Mol Cell Biol 11:621-632, 2010). Therefore, the measurement of Cyt C release can give vital information about cell death signaling. Immunolabeling against Cyt C can give an easy readout of mitochondrial integrity as well, allowing for simultaneous identification of mitochondrial viability (and/or damage) and initiation of intracellular death processes. In this chapter, we use Cyt C as a dual marker of mitochondrial integrity and cell death and review several protocols to measure Cyt C localization into intact mitochondria and its release into the cytosol. The goal is to offer an array of assays that, combined, provide both qualitative and quantitative analysis of the relationship between mitochondrial viability and activation of an intracellular cell death process. Immunofluorescence, Western blot, and ELISA measurements of Cyt C as are discussed in detail.

ALS-linked mutant SOD1 damages mitochondria by promoting conformational changes in Bcl-2.

In mutant superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS), accumulation of misfolded mutant SOD1 in spinal cord mitochondria is thought to cause mitochondrial dysfunction. Whether mutant SOD1 is toxic per se or whether it damages the mitochondria through interactions with other mitochondrial proteins is not known. We previously identified Bcl-2 as an interacting partner of mutant SOD1 specifically in spinal cord, but not in liver, mitochondria of SOD1 mice and patients. We now show that mutant SOD1 toxicity relies on this interaction. Mutant SOD1 induces mitochondrial morphological changes and compromises mitochondrial membrane integrity leading to release of Cytochrome C only in the presence of Bcl-2. In cells, mouse and human spinal cord with SOD1 mutations, the binding to mutant SOD1 triggers a conformational change in Bcl-2 that results in the uncovering of its toxic BH3 domain and conversion of Bcl-2 into a toxic protein. Bcl-2 carrying a mutagenized, non-toxic BH3 domain fails to support mutant SOD1 mitochondrial toxicity. The identification of Bcl-2 as a specific target and active partner in mutant SOD1 mitochondrial toxicity suggests new therapeutic strategies to inhibit the formation of the toxic mutant SOD1/Bcl-2 complex and to prevent mitochondrial damage in ALS.