SPORADIC AMYOTROPHIC LATERAL SCLEROSIS IN PATIENTS WITH COMMON GEOGRAPHICAL ORIGIN: A MULTIDISCIPLINARY STUDY

Amyotrophic Lateral Sclerosis (ALS) is a late onset, fatal, neurodegenerative disorder that selectively affects motor neurons. It leads to the degeneration of both upper and lower motor neurons, respectively in the motor cortex and in the brainstem and spinal cord. Different mechanisms have been proposed to explain the pathogenesis of the disease: protein aggregation, oxidative stress, impairment of mitochondrial function, transcription dysfunctions, alterations in the proteasome pathway, inflammation and excitotoxicity. A wide phenotypical variability is described, likely attributable to a combination of genetic and environmental factors, able to modify the clinical expression of the disease. However, the difficulty to focus on one specific environmental factor, the variability of such exposure in space and time and the interaction between environment and genetic background, hampered the evaluation of their possible role in ALS etiology. The study of geographical clusters of ALS patients has been helpful - in the past years - to get more insights into the pathology. Studying individuals exposed to the same environment, thus ideally subjected to the same exogenous stressors, could be very valuable by limiting one of the most confounding variables in ALS studies. Indeed, a diverse environmental exposure is almost always necessarily present when analyzing the large cohorts of patients that are usually required to reach statistically significant numbers of cases in epidemiological-type studies. This PhD thesis presents a multidisciplinary study performed on a small cohort of sporadic ALS patients, all originating from a restricted and defined geographical area. Focusing on a very limited geographical area, gave the chance to consider the characteristics of the surrounding environment and allowed to raise hypotheses on the possibly involved stressors acting on the local population, being those environmental or dietary contributions. In detail the experiments performed can be divided into five main themes: • To assess possible differences in the diet of ALS subjects and healthy controls and to provide information as for whether the consumption of one or more foods were associated to the disease status by an exploratory questionnaire submitted to the subjects involved in the study. There were no evident differences in the nutrition habits of ALS subjects with respect to healthy controls. • To determine concentrations of a selected panel of metals in serum and whole blood with ICP-MS. Metals analyzed have been chosen based on their biological relevance and previous works. Concentrations of As, Al, Mn, Se, Ni, Pb, Hg, Cu, Fe, Zn, Co, Cr, Ba, Sn, U, V, Sr have been evaluated in serum, while an additional analysis of Cr, Mn, Co, Cu, Zn, As, Se, Pb and Hg has been performed on whole blood. The most striking result comes from the association of lower levels of serum As with the disease status, an occurrence reported for the first time. • To analyze the serum proteome for the first time in ALS studies, through two-dimensional electrophoresis, to dissect the possible links between circulating proteins and circulating metals and to exploit this technique to look for a possible panel of easily accessible disease biomarkers. Proteins in which was registered an alteration are involved in the Acute Phase Response. Indeed, the different expression with respect to controls could be related to the disease status of the subjects. Alterations in some proteins related to lipid homeostasis have been detected, that is consistent with a proposed metabolic shift towards an increased peripheral use of lipids in ALS. Deregulation of lipid homeostasis proteins seems to be more directly linked in modulating the disease progression, as supported from literature data. • To evaluate the genetic background of patients by analyzing the most frequently mutated genes (SOD1, FUS, TDP43 and C9orf72) to exclude a genetic cause of the disease, giving more relevance to the environmental exposure as a risk factor. APOE4 and PON genotypes have been evaluated in the light of the results obtained from the proteomic studies. The allelic frequency for the APOE*4 allele, associated to neurodegeneration, is 3-fold higher as well as APOE*3/APOE*4 genotype in the patients’ group than in control’s. • To evaluate the DNA oxidative stress by Comet Assay, since it is well known that cellular oxidative stress is involved in the disease. Furthermore, metals impaired homeostasis may exacerbate oxidative stress via Fenton reactions. In literature at present there are few works evaluating this aspect with this approach. The levels of endogenous DNA damage did not differ between ALS and control group. To overcome the obvious limits related to the small number of subjects necessarily involved in this study, advantage was taken from the application of a multifactorial statistical evaluation, based on a machine learning software. This software, belonging to the artificial neural networks architecture, was specifically designed to analyze complex problems, where the number of variables significantly exceeds the amount of subjects involved in the study, as usually is in the case of rare diseases. Great amount of data is, nowadays, a limit in understanding results of experiments performed with more and more sophisticated technologies, especially in complex diseases such as ALS. The application of new statistical analyses, based on machine learning, where data are the basis of the creation of models to interpret interactions among variables, would be the key to translate raw data into understandable models. The lack of an effective pharmacological treatment in ALS leads to rapidly looking for alternative approaches to modulate this devastating disease. To this purpose, a nutritional intervention, with the development of specific nutritional formulas, would be an effective tool to intervene on the patients’ lipid profile and to contrast potential metal’s homeostasis impairment. Thus, development of such formulations is strongly supported from the evidences raised from this study, together with its application in clinical trials.