Efeito da inativação da superóxido dismutase 1 humana para a desregulação da glicólise aeróbica e seu impacto no desenvolvimento da esclerose lateral amiotrófica

Abstract

Amyotrophic lateral sclerosis (ALS) causes the degeneration of motor neurons and associated glial cells. Astrocytes perform numerous neuron support functions, such as metabolic cooperation. Astrocytes produce lactate, which is sent to neurons for energy production through the oxidative phosphorylation pathway. Thus, neurons spare glucose for the production of NADPH, which helps protect against oxidative stress and prevent the generation of toxic by-products of glycolysis, such as the glycating agent methylglyoxal (MGO), whose elimination is deficient in neurons. Lactate production in astrocytes occurs through aerobic glycolysis, a metabolic process in which cells ferment even in the presence of oxygen. This occurs because the enzyme pyruvate dehydrogenase (PDH) is phosphorylated and therefore inhibited. Several changes have been observed in astrocytes in ALS patients, some of which may be directly related to this metabolic cooperation, highlighting the importance of better understanding its mechanism and regulation. A possible candidate for signalling PDH phosphorylation would be Cu/Zn superoxide dismutase (SOD1). Among its numerous functions, SOD1 has been shown to participate in the regulation of aerobic glycolysis in the yeast Saccharomyces cerevisiae. Because of this, and the association between SOD1 and ALS in familial and sporadic cases, this study aims to analyze the ability of human SOD1 to regulate aerobic glycolysis and investigate the possible impact of ALS-related alterations on this process. In a yeast model, where endogenous SOD1 was replaced by the human ortholog, the WT human protein was found to regulate aerobic glycolysis in the same manner as yeast, unlike ALS-associated SOD1 mutants and SOD1-deficient yeast. The effect of human SOD1 on the regulation of aerobic glycolysis was also found to involve the enzyme's activity level and PDH phosphorylation. In this work, we analyzed the effect of SOD1 on the regulation of aerobic glycolysis using three distinct approaches: the effect of SOD1 deficiency using the HEK293 knockout (KO) strain; the effect of SOD1 damage by stressing HEK293 WT with MGO; and the effect of a SOD1 mutation using primary cultures of astrocytes obtained from transgenic mice expressing the G93A mutant human SOD1, found in ALS patients. According to the results, both in HEK KO SOD1 and in astrocytes from transgenic mice expressing mutant human SOD1, a decrease in fermentative capacity was observed (increased oxygen consumption and decreased rates of lactate production and glucose consumption). This was associated with a decrease in the level of phosphorylated PDH (and an increase in PDH activity in the HEK model), corroborating the results found in the yeast model. HEK293 WT cells stressed with MGO showed increased PDH activity and decreased SOD1 activity; however, no metabolic changes were observed. Thus, using different models—yeast, human cell lines, and primary mouse astrocyte cultures—we can conclude that deficiencies in human SOD1 (lack or mutations) impair the regulation of aerobic glycolysis, which may be related to the development of ALS.