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Canine degenerative myelopathy (DM) is a human amyotrophic lateral sclerosis (ALS)-like neurodegenerative disease. It is a unique, naturally occurring animal model of human ALS. Canine DM is associated with the aggregation of canine superoxide dismutase 1 (cSOD1), which is similar to human ALS. Almost 100% of cases in dogs are familial, and the E40K mutation in cSOD1 is a major causative mutation of DM. Therefore, it is important to understand the molecular mechanisms underlying cSOD1(E40K) aggregation. To address this, we first analyzed the structural model of wild type cSOD1. Interactions were evident between amino acid E40 and K91. Therefore, the mutation at residue E40 causes loss of the interaction and may destabilize the native structure of cSOD1. Differential scanning fluorimetry revealed that the E40K mutant was less stable than the wild type. Moreover, stability could be recovered by the E40K and K91E double mutation. Acceleration of amyloid fibril formation in vitro and aggregate formation in cells of cSOD1(E40K) was also suppressed by the introduction of this double mutation in thioflavin T fluorescence assay results and in transfectant cells, respectively. These results clearly show the importance of the interaction between amino acid residues E40 and K91 in cSOD1 for the stability of the native structure and aggregation.

Canine degenerative myelopathy (DM) is a progressive neurodegenerative disorder, which is commonly associated with c.118G > A (p. E40K) missense mutation in the superoxide dismutase 1 (SOD1) gene. Mutant SOD1 protein (SOD1 E40K ) is likely to be misfolded, acquire insolubility, aggregate in the cytoplasm of neural cells, and lead to degeneration of the nervous tissues. Along with a chaperone activity, macrophage migration inhibitory factor (MIF) is a multifunctional protein that has been shown to directly inhibit human mutant SOD1 misfolding and enhance survival of mutant SOD1-expressing motor neurons. The purpose of this study was to determine whether MIF also inhibits DM-related SOD1 E40K misfolding and accumulation of SOD1 aggregates. Human embryonic kidney 293A cells were transfected SOD1 cWT or SOD1 E40K with or without MIF. The percentages of cells containing transfected SOD1 aggregates were measured by immunocytochemistry, and the amount of SOD1 E40K in the insoluble fraction was evaluated by immunoblotting. The percentage of cells with SOD1 E40K aggregates and the amount of insoluble SOD1 E40K protein decreased in the presence of MIF. Because the chaperone activity of MIF assists in SOD1 E40K folding and enhances the refolding and degradation of misfolded SOD1 E40K , the results of this study suggests that MIF regulates the accumulation of SOD1 aggregates by its chaperone activity. We propose that enhancing intracellular MIF chaperone activity could be an effective therapeutic strategy for DM., (Copyright © 2022. Published by Elsevier Ltd.)

Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on amyloidogenic protein aggregation. However, no effective drug suitable for transition to clinical use has been found. Here we examined several novel oxindole compounds (GIF compounds) for their inhibitory effects on aggregate formation of the canine mutant superoxide dismutase 1 (cSOD1 E40K), a causative mutation resulting in DM, using Thioflavin-T fluorescence. Most GIF compounds inhibited the aggregation of cSOD1 E40K. Among the compounds, GIF-0854-r and GIF-0890-r were most effective. Their inhibitory effects were also observed in cSOD1 E40K-transfected cells. Additionally, GIF-0890-r effectively inhibited the aggregate formation of human SOD1 G93A, a causative mutation of ALS. GIF-0827-r and GIF-0856-r also effectively inhibited aggregate formation of human prion protein (hPrP). Subsequently, the correlation between their inhibitory effects on cSOD1 and hPrP aggregation was shown, indicating GIF compounds inhibited the aggregate formation of multiple amyloidogenic proteins. Conclusively, the novel oxindole compounds (GIF-0827-r, GIF-0854-r, GIF-0856-r, and GIF-0890-r) are proposed as useful therapeutic candidates for amyloidogenic neurodegenerative disorders., (Copyright © 2022. Published by Elsevier B.V.)

Canine degenerative myelopathy (DM) is an adult-onset progressive and fatal neurodegenerative disorder. Superoxide dismutase 1 (SOD1) mutations have been reported in affected dogs and immunohistochemical analyses revealed the accumulation of mutant SOD1 (E40K) in spinal neurons and astrocytes. Therefore, this disease is regarded as a unique spontaneous large-animal model of SOD1-mediated amyotrophic lateral sclerosis (ALS) in humans. Recent studies reported that endoplasmic reticulum (ER) stress is a key pathomechanism underlying motor neuron death in ALS. The present study demonstrated the up-regulated expression of the ER stress marker GRP78/BiP (BiP) in the spinal cords of DM-affected dogs. Immunohistochemistry of serial spinal cord sections revealed strong BiP expression in microglia and astrocytes in DM compared to normal control dogs, whereas such difference was not observed in spinal neurons. The results of transcriptional analyses of DM spinal tissues showed increased expression levels of apoptosis signal-regulating kinase 1 (ASK1) and spliced X-box binding protein (XBP1s). E40K-transfected Neuro2A cells expressed higher levels of BiP than wild-type SOD1-transfected cells. These results suggest that the activation of the unfolded protein response (UPR) in microglia and astrocytes plays crucial roles in UPR-mediated inflammation in the spinal cords of DM-affected dogs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Canine degenerative myelopathy (DM) is a fatal neurodegenerative disorder. Dogs with typical clinical signs carry homozygous mutations in the superoxide dismutase 1 (SOD1) gene; therefore, DM is regarded as a naturally-occurring model of amyotrophic lateral sclerosis (ALS). Despite the presence of a toxic mutant protein, E40K-SOD1 heterozygotes rarely develop clinical signs. Therefore, E40K-heterozygotes may provide insights into the subclinical and early phase of mutant SOD1-related pathology. In order to identify the distribution of mutant SOD1 in the spinal cords of E40K-heterozygotes, we developed a monoclonal antibody 16G9 that reacts to the mutant E40K-SOD1 protein. We found that the spinal cords of E40K-heterozygotes display white matter degeneration, the severity of which was markedly less than that in E40K-homozygotes. In E40K-heterozygotes, 16G9-reactive SOD1 accumulated predominantly in reactive astrocytes, while spinal neurons remained almost completely free of this form of SOD1 proteins. In contrast, all symptomatic E40K-homozygotes contained 16G9-reactive SOD1 in their spinal neurons and reactive astrocytes. These results suggest that mutant SOD1 proteins accumulate in reactive astrocytes during the early phase of DM pathology, which may contribute to subclinical neurodegeneration. The early involvement of reactive astrocytes in the pathogenesis of DM is strongly suspected and warrants further investigations in the context of non-cell autonomous neuronal death, as proposed for ALS., (Copyright © 2016 Elsevier B.V. All rights reserved.)

• Canine mutant superoxide dismutase-1 (SOD1; E40K SOD1) proteins formed aggregates. • E40K SOD1 aggregates were released from cells and taken up by other cells. • E40K SOD1 aggregates induced aggregate formation of natively fold SOD1 proteins. • Cell to cell E40K SOD1 aggregate propagation could underpin degenerative myelopathy. Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate formation is largely unknown. In human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), cell-to-cell propagation of disease-relevant proteins has been demonstrated. Therefore, in this study, propagation of aggregation-forming property of mutant SOD1 protein in DM in vitro was investigated. This study demonstrated that aggregates composed of canine wild type SOD1 protein were increased by co-transfection with canine mutant SOD1 (E40K SOD1), indicating intracellular propagation of SOD1 aggregates. Further, aggregated recombinant SOD1 proteins were released from the cells, taken up by other cells, and induced further aggregate formation of normally folded SOD1 proteins. These results suggest intercellular propagation of SOD1 aggregates. The hypothesis of cell-to-cell propagation of SOD1 aggregates proposed in this study may underly the progressive nature of DM pathology. [ABSTRACT FROM AUTHOR]