Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder which is characterized by the progressive loss of both cortical and spinal motor neurons (MN). In Europe, ALS is found in approximately 1 in every 50,000 members of the population and there is a cumulative lifetime risk of its development of 1 in every 400 people [1, 2]. Cases are inherited ALS (familial ALS, or FALS) are relatively infrequent (fewer than 10%), with only 20% of these being attributable to a single, specific molecular defect [3]. Although ALS is generally fatal amongst such patients, resulting in death within a period of 2–4 years, survival times vary considerably for individual patients [4]. The aetiology of the majority of ALS cases (the remaining 90%, which are classified as sporadic ALS, or SALS) remains unknown, but oxidative stress is generally thought to be involved and to play an important role in in causing motor neuron death [5]. Mitochondrial degeneration and protein aggregation mediated by oxidative stress also appear to play major roles in associated progressive motor neuron death [6, 7]. Mitochondrial proteins linked to energy production have been identified as key targets for oxidative stress; this is logical given that mitochondria are the main intracellular source of free radicals[8]. In consequence, it is widely held that free radical-dependent mitochondrial damage plays a fundamental role in neurodegeneration associated with ALS [9, 10].
Diagnosis often occurs only during the later stages of the disease when, unfortunately, around 50% of the motor neurons have already been lost [11]. This problem is exacerbated by the fact that only two drugs have been approved for use in the treatment of ALS: riluzole and edaravone and that they confer only limited benefits in terms of restricting the progression of the disease [12].
Until another more reliable diagnostic test for ALS is developed, it is possible to use biomarkers as tools for early diagnosis, predictors of prognoses, and indicators of target engagement and/or therapeutic responses; they may also aid in the discovery and development of future therapeutic treatments for ALS [13, 14]. It may be possible to find an efficient therapeutic strategy by improving our knowledge of energy metabolism-related molecules, including that of Aco2 [15].
Several ALS-related biological biomarkers have been identified in recent decades, some of which are related to blood [16–19]. Knowing that ALS is a multisystemic disease, identifying a panel of biomarkers capable of accurately reflecting features of the pathology would become an interesting priority. This would not only serve for diagnostic purposes, but also for prognostic and predictive applications [20].
Mitochondrial Aconitase, also known as Aconitase 2 (Aco2), is a key Krebs cycle enzyme that contains an iron-sulphur cluster which is highly sensitive to oxidative damage[21]. Aco2 can be found in the mitochondrial matrix and plays a role in the generation of energy. It can, however, be susceptible to high levels of oxidative stress and this may cause its inactivation [21]. Aconitase is a protein that is known to be susceptible to oxidative modification and which may suffer inactivation as a result of aging and/or various oxidative stress-related disorders [22]. This enzyme plays a role in the regulation of cellular metabolism and in iron homeostasis, as it balances the regulatory, and damaging, effects of reactive oxygen species (ROS) [21]. Studies conducted in animal and cellular models have shown that free radicals and the products of oxidative damage can promote the oxidation of Aco2, with this resulting in impaired enzymatic activity [23]. This may subsequently result in Aco2 aggregating and accumulating in the mitochondrial matrix and causing mitochondrial dysfunction [23]. There is growing evidence to suggest a direct association between impaired energy metabolism and the incidence and progression of neurodegenerative diseases [23]. It has already been established that changes in certain bioenergetic parameters are often associated with pathological features of neurodegenerative diseases and can produce neuronal dysfunction [22].
An observed loss of Aco2 activity may therefore point to increased levels of mitochondrial dysfunction as a result of oxidative damage, which could be relevant to ALS pathogenesis.
Our study looked to confirm changes in Aco2 activity and to determine whether such changes were dependent on, or independent of, the patient's condition. We also sought to explore the feasibility of using these changes as biomarkers, with which to quantify disease progression, and also as predictors for prognosis. We analysed the correlation between mitochondrial antioxidant activity and several different variables that were relevant to the clinical evolution of the disease (age, time from onset, bulbar or spinal onset, initial upper or lower MN signs). We also examined severity criteria (nutritional and respiratory status), ALS functional rating scores (ALSFRS) [24–26], and prognostic factors - such as lipid [27, 28] and ferritin levels [29, 30]. These were studied in a series of ALS patients at different disease stages.