Multidisciplinary study of sALS in patients originating from a restricted geographical area

Amyotrophic Lateral Sclerosis (ALS) is a rare neurodegenerative disorder characterized by selective degeneration of both upper and lower motor neurons in the brain, brainstem, and spinal cord. This results in paralysis due to muscle weakness and atrophy, leading to death in 3-5 years1. Genetic and environmental factors are involved in the pathogenesis of this disease and metals metabolism has been linked to ALS2. Proteomic studies are currently being performed to search for possible biomarkers3. Here we present a study aimed at investigating different aspects of the disease, based on a multidisciplinary approach. The cohort of ALS patients that we analyzed includes seven patients, all originating from a common, restricted, geographical area and five matched controls. Environmental exposure is the same for all these subjects. SOD1, FUS, TDP43, C9ORF72 and APOE genotypes were evaluated. For metal quantitation, samples of serum and whole blood were analyzed by ICP-MS. For proteomic analyses, immobilized pH gradient covered the 4-10 and 3-7 pH range both in reducing and non-reducing conditions. Levels of DNA oxidation were evaluated by a comet assay. Statistical analyses were carried out with Student’s t-test and Artificial Neural Networks. Among the metals analyzed in serum, As concentration resulted significantly lower in patients than in controls (p=0.007); Mn and Hg showed lower levels in patients. Auto-CM analysis linked closely high concentrations of Al and Se to the ALS group. Levels of metals in whole blood have been correlated with levels in serum. Our proteomics data show that some proteins related to Acute Phase Response (APR) and lipid homeostasis are decreased in patients (APOA1, APOA2, TTR, RET4 and SAP) while only ANT3 results increased. For some of these proteins we can describe a drastic reduction in the first 5 years of disease. Apoε4 allele is more represented in the patient’s group than in controls’. Impaired metal homeostasis, attributable to environmental exposure, could lead to mineral overload. Waters of the creek of the narrow valley where these subjects are located, are reported to be strongly polluted due to Acid Mine Drainage. Besides promoting oxidative stress, metals can compete for the binding sites of metal-containing proteins, such as those containing iron-sulfur clusters4. The different expression of the APR proteins reported could be a reflection of the disease status of the subjects analyzed, possibly linking ALS to a chronic inflammation status. Enrichment in Apoε4 allele frequency in patients may provide a link between neurodegeneration and lipid metabolism disturbances. It is important to highlight the fact that all the proteins found differentially expressed in our study have already been described in other studies. This strengthens our methodological approach, based on a small number of patients but with a common environmental exposure. References. 1 Chiò et al. (2013) Global epidemiology of amyotrophiclateral sclerosis: a systematic review of the published literature. Neuroepidemiology 2 Hadzhieva et al. (2014) Review: iron metabolism and the role of iron in neurodegenerative disorders. Neuropathol Appl Neurobiol 3 Krüger et al. (2013) Proteome analysis of body fluids for amyotrophic lateral sclerosis biomarker discovery. Proteomics - Clinical Applications 4 De Benedetti et al. (2016) Serum metal evaluation in a small cohort of Amyotrophic Lateral Sclerosis patients reveals high levels of thiophylic species. Peptidomics