Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Bioenergetics in Amyotrophic Lateral Sclerosis, new experimental approaches and therapeutic potentials

Background and Purpose Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease, is considered the prototype of motor neuron disease, characterized by motor neuron loss, muscle waste and paralysis of voluntary muscles is currently incurable despite intense research and numerous unsuccessful clinical trials. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. Although considered as a pure motor neuron disease, increasing evidence indicates that the sole protection of motor neurons by a single target drug is not sufficient to improve the pathological phenotype. In fact, so far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We therefore evaluated the therapeutic potential of the multi-target drug, TMZ, in SOD1G93A mice. Experimental Approach TMZ is an anti-ischemic drug used for the treatment of coronary artery disease. As a metabolic modulator, TMZ improves glucose metabolism. Furthermore, TMZ enhances mitochondrial metabolism and promotes nerve regeneration, exerting an anti-inflammatory and antioxidant effect. Here, we orally treated SOD1G93A mice with TMZ, solubilized in drinking water at a dose of 20 mg/kg, from disease onset. We assessed the impact of TMZ on disease progression by studying metabolic parameters, grip strength, and histological alterations in skeletal muscle, peripheral nerve and spinal cord. We also addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. Key Results TMZ administration delays motor function decline, improves muscle performance and metabolism, and significantly extends overall survival of SOD1G93A mice (increased median survival of 16 days and 12.5 days for male and female respectively). Moreover, TMZ prevents the dismantlement of neuromuscular junctions, attenuates motor neuron loss and reduces neuroinflammation in the spinal cord and in peripheral nerves. We have also characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-patient-derived peripheral blood mononuclear cells (PBMCs). Conclusion and Implications In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. In SOD1G93A mice, therapeutic effect of TMZ is underpinned by its action on mitochondrial function in skeletal muscle and spinal cord. Furthermore, the findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.