Riassunto analitico
Amyotrophic lateral sclerosis (ALS) is the most prevalent type of motoneuron disorder, characterized by a progressive deterioration of both upper and lower motor neurons, causing muscle wasting, gradual paralysis, and ultimately death. The average survival from diagnosis is 2–3 years. Approximately 10% of ALS cases follow an autosomal dominant inheritance pattern, while the remaining 90% of cases appear to have no clear genetic basis. Mutations in multiple genes have been identified in ALS, including superoxide dismutase 1 (SOD1), Fused in sarcoma (FUS), TAR DNA binding protein (TARDBP; TDP-43), and C9orf72. Mitochondrial impairment is a common characteristic of several neurodegenerative diseases, including ALS. Alterations of mitochondrial morphology, dynamics, metabolic activity, oxidative phosphorylation, and production of reactive oxygen species (ROS) has been observed in ALS patients. Moreover, many of the genes associated with ALS are involved in mitochondrial-associated functions. In the present thesis we report a functional analysis of mitochondria and the impact of oxidative stress in different study groups of patients with ALS. As mitochondrial DNA (mtDNA) is released in the extracellular space after cell damage and death, we measured the mtDNA amount in plasma of 40 patients with ALS and 20 healthy controls, to determine if this parameter could be correlated with motoneuron damage. MtDNA levels increase with aging and ALS patients had a lower mtDNA plasma level if compared to healthy, matched controls. However, when stratified by sex and age, we observed that this phenomenon was mainly due to the difference observed in middle aged patients, particularly in female, who present a lower level of mtDNA than control. Then, we focused our attention on the alterations of mitochondria in cells from ALS patients, which could contribute to the release of mtDNA from mitochondria to the cytosol and/or in the extracellular space. We analyzed mitochondria in primary culture of fibroblasts from a male patient bearing the pathogenic mutation pA382T in TDP-43, and from two female discordant monozygotic twins, one diagnosed with ALS and carried a mutation in the progranulin gene, while the other twin was healthy. Fibroblasts from patient carrying pA382T mutation were characterized by profound alterations of cell proteome, which impacts particularly the mitochondrial metabolic pathways. TDP-43 levels were similar to control fibroblasts, but a higher fraction localized in mitochondria. Mitochondrial network was fragmented, and the organelles smaller and more spheric. In agreement with impaired proteome and mitochondrial morphology, basal cell respiration was reduced. MtDNA levels appeared normal, but a higher amount of mtDNA was present in the cytosol, suggesting a pronounced mtDNA misplacement which can promote a proinflammatory response. Concerning discordant twins, fibroblasts from the affected twin showed a slower growth rate and morphological differences compared to those from the healthy twin. Similar to what observed in fibroblasts from TDP-43 patient, mitochondrial network appeared fragmented, and the organelles smaller and more spheric in fibroblasts from ALS twin. Nevertheless, higher levels of ROS, higher basal and maximal respiration, and higher mitochondrial membrane potential was observed in fibroblasts from the affected twin, in partial contrast with what observed in the patient with TDP-43 mutation. In conclusion, this data confirms that mitochondria from ALS patients displays a wide range of functional and morphological alterations, but with a high degree of heterogeneity, indicating that the mechanistic links mitochondrial impairment and ALS pathogenesis greatly vary on the basis of the genetic alterations.
|