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Cu/Zn superoxide dismutase (SOD1) mutations are associated with amyotrophic lateral sclerosis (ALS). SOD1-positive aggregates in motor neurons, as well as proteins that interact with the aggregates are presumably involved in ALS neurotoxicity. We used a proteomics approach to compare differences in protein expression in spinal cord homogenates from non-transgenic (NTG) and ALS model mice. Using the homogenates, we identified proteins that interacted with SOD1 seeds in vitro. We assessed differences in SOD1-interacting proteins in cell cultures treated with proteasome or autophagy inhibitor. In the first experiment, intermediate filamentous and small heat shock proteins were upregulated in glial cells. We identified 26 protein types that interacted with aggregation cores in ALS model homogenates, and unexpectedly, 40 proteins in were detected in NTG mice. In cell cultures treated with proteasome and autophagy inhibitors, we identified 16 and 11 SOD1-interacting proteins, respectively, and seven proteins in untreated cells. These SOD1-interacting proteins were involved in multiple cellular functions such as protein quality control, cytoskeletal organization, and pathways involved in growth factor signaling and their downstream cascades. The complex interactions between pathways could cause further dysregulation, ultimately leading to fatal cellular dysfunction in ALS.

Heat shock proteins play roles in assisting other proteins to fold correctly and in preventing the aggregation and accumulation of proteins in misfolded conformations. However, the process of aging significantly degrades this ability to maintain protein homeostasis. Consequently, proteins with incorrect conformations are prone to aggregate and accumulate in cells, and this aberrant aggregation of misfolded proteins may trigger various neurodegenerative diseases, such as Parkinson&rsquo, s disease. Here, we investigated the possibilities of suppressing &alpha, synuclein aggregation by using a mutant form of human chaperonin Hsp60, and a derivative of the isolated apical domain of Hsp60 (Hsp60 AD(Cys)). In vitro measurements were used to detect the effects of chaperonin on amyloid fibril formation, and interactions between Hsp60 proteins and &alpha, synuclein were probed by quartz crystal microbalance analysis. The ability of Hsp60 AD(Cys) to suppress &alpha, synuclein intracellular aggregation and cytotoxicity was also demonstrated. We show that Hsp60 mutant and Hsp60 AD(Cys) both effectively suppress &alpha, synuclein amyloid fibril formation, and also demonstrate for the first time the ability of Hsp60 AD(Cys) to function as a mini-chaperone inside cells. These results highlight the possibility of using Hsp60 AD as a method of prevention and treatment of neurodegenerative diseases.