Coactivation of GSK3β and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients’ MNs, SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1G85R and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1G93A ALS mice. Moreover, we found that coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3β activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3β and IGF-1 activation) that may serve as potential targets for ALS therapy.

A Natural History Comparison of SOD1-Mutant Patients with Amyotrophic Lateral Sclerosis between Chinese and German Populations

Background: The gene coding the Cu/Zn superoxide dismutase ( SOD1 ) was the first-identified causative gene of amyotrophic lateral sclerosis (ALS), and the second most common genetic cause for ALS worldwide. The promising therapeutic approaches targeting SOD1 mutations are on the road. The purpose of the present study was to compare the mutational and clinical features of Chinese and German patients with ALS carrying mutations in SOD1 gene, which will facilitate the strategy and design of SOD1 -targeted trials.Methods: Demographic and clinical characteristics were collected from two longitudinal cohorts in China and Germany. Chinese and German patients carrying SOD1 mutations were compared with regard to mutational distribution, age of onset, site of onset, body mass index (BMI) at diagnosis, diagnostic delay, progression rate, and survival.Results: A total of 66 Chinese and 84 German patients with 69 distinct SOD1 mutations were identified. The most common mutation in both populations was p.His47Arg. It was found in 8 Chinese and 2 German patients and consistently showed a slow progression of disease in both countries. Across all mutations, Chinese patients showed a younger age of onset (43.9 vs 49.9 years, p=0.002), a higher proportion of young-onset cases (62.5% vs 30.7%, p<0.001) and a lower BMI at diagnosis (22.8 vs 26.0, p<0.001) compared to German patients. Although riluzole intake was less frequent in Chinese patients (28.3% vs 81.3%, p<0.001), no difference in survival between populations was observed (p=0.90). Across both cohorts, female patients had a longer diagnostic delay (15.0 vs 11.0 months, p=0.01) and a prolonged survival (248.0 vs 60.0 months, p=0.005) compared to male patients.Conclusions: Our data demonstrate the distinct mutational and clinical spectrums of SOD1 -mutant patients in Asian and European populations. Clinical phenotypes seem to be primarily influenced by mutation-specific, albeit not excluding ethnicity-specific factors. Further large-scale transethnical studies are needed to clarify determinants and modifiers of SOD1 phenotypes.

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans

Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

Copper-ATSM as a Treatment for ALS: Support from Mutant SOD1 Models and Beyond

The blood–brain barrier permeant, copper-containing compound, CuII(atsm), has successfully progressed from fundamental research outcomes in the laboratory through to phase 2/3 clinical assessment in patients with the highly aggressive and fatal neurodegenerative condition of amyotrophic lateral sclerosis (ALS). The most compelling outcomes to date to indicate potential for disease-modification have come from pre-clinical studies utilising mouse models that involve transgenic expression of mutated superoxide dismutase 1 (SOD1). Mutant SOD1 mice provide a very robust mammalian model of ALS with high validity, but mutations in SOD1 account for only a small percentage of ALS cases in the clinic, with the preponderant amount of cases being sporadic and of unknown aetiology. As per other putative drugs for ALS developed and tested primarily in mutant SOD1 mice, this raises important questions about the pertinence of CuII(atsm) to broader clinical translation. This review highlights some of the challenges associated with the clinical translation of new treatment options for ALS. It then provides a brief account of pre-clinical outcomes for CuII(atsm) in SOD1 mouse models of ALS, followed by an outline of additional studies which report positive outcomes for CuII(atsm) when assessed in cell and mouse models of neurodegeneration which do not involve mutant SOD1. Clinical evidence for CuII(atsm) selectively targeting affected regions of the CNS in patients is also presented. Overall, this review summarises the existing evidence which indicates why clinical relevance of CuII(atsm) likely extends beyond the context of cases of ALS caused by mutant SOD1.

Pro-Oxidant Activity of an ALS-Linked SOD1 Mutant in Zn-Deficient Form

Cu, Zn superoxide dismutase (SOD1) is a representative antioxidant enzyme that catalyzes dismutation of reactive oxygen species in cells. However, (E,E)-SOD1 mutants in which both copper and zinc ions were deleted exhibit pro-oxidant activity, contrary to their antioxidant nature, at physiological temperatures, following denaturation and subsequent recombination of Cu2+. This oxidative property is likely related to the pathogenesis of amyotrophic lateral sclerosis (ALS); however, the mechanism by which Cu2+ re-binds to the denatured (E,E)-SOD1 has not been elucidated, since the concentration of free copper ions in cells is almost zero. In this study, we prepared the (Cu,E) form in which only a zinc ion was deleted using ALS-linked mutant H43R (His43→Arg) and found that (Cu,E)-H43R showed an increase in the pro-oxidant activity even at physiological temperature. The increase in the pro-oxidant activity of (Cu,E)-H43R was also observed in solution mimicking intracellular environment and at high temperature. These results suggest that the zinc-deficient (Cu,E) form can contribute to oxidative stress in cells, and that the formation of (E,E)-SOD1 together with the subsequent Cu2+ rebinding is not necessary for the acquisition of the pro-oxidant activity.

Comprehensive identification of HSP70/HSC70 Chaperone Clients in Human Cells

The HSP70 family of chaperones are the front-line of protection from stress-induced misfolding and aggregation of polypeptides in most organisms and are responsible for promoting the stability, folding, and degradation of clients to maintain cellular protein homeostasis. Here we demonstrate quantitative identification of HSP70 and HSC70 clients using an ubiquitin-mediated proximity tagging strategy and show that, despite their high degree of similarity, these enzymes have largely non-overlapping specificities. Both proteins show a preference for association with newly synthesized polypeptides but each responds differently to changes in the stoichiometry of proteins in obligate multi-subunit complexes. In addition, expression of an ALS-associated SOD1 mutant protein induces changes in HSP70 and HSC70 client association and aggregation toward polypeptides with predicted disorder, indicating that there are global effects from a single misfolded protein that extend to many clients within chaperone networks. Together these findings show that the ubiquitin-mediated UBAIT fusion system can efficiently isolate the complex interactome of HSP chaperone family proteins under normal and stress conditions.