ASSESSMENT OF CELL AND METABOLIC IMPACT OF MICROTUBULE TARGETING AGENTS (MTA) IN NEURODEGENERATION AND NEURON MODELS.

Parkinson’s disease (PD) is primarily characterized by its sporadic onset, with 80–85% of cases occurring without a clear genetic cause, classified as idiopathic. The complexity of PD’s pathogenesis makes it challenging to identify a single molecular mechanism responsible for the disease. Rather, PD progression is associated with a range of pathological processes, including mitochondrial dysfunction, oxidative stress, disturbances in cytoskeletal dynamics, abnormal protein aggregation, and neuroinflammation. Current treatment options for PD focus mainly on alleviating motor symptoms through dopamine replacement therapies, such as levodopa and dopamine agonists.While these treatments can provide temporary relief, they do not address the underlying neurodegeneration and typically lose effectiveness over time. Additionally, long-term use can lead to complications such as motor fluctuations and dyskinesias. To date, no disease-modifying therapies have been proven to be effective in ameliorating the progression of PD, underscoring the need for new therapeutic strategies. In this context, in this work we investigated some of the pathological mechanisms involved in PD, including its onset, progression, and potential treatment strategies, in order to unveil the pathophysiological pathways shaping the disease and to provide possible new insights for therapeutic intervention. Based on the metabolic characterization and transcriptomic data, and applying specific metabolic reconstructions after data integration using Genome-Scale Metabolic Models (GSMM) we report that, in PC12 cell model, neuronal differentiation is accompanied by switching towards a more glycolytic metabolism, with the glucose catabolism that tends towards lactate production, rather than the aerobic oxidative pathway, with a general reduction of TCA and OXPHOS subsystems. Despite surprising, this may depend on the signaling properties of lactate in promoting neuronal differentiation. In addition, we observe an overall reduction in amino acids consumption upon differentiation, probably due to the increased mitochondrial fission, which may fit the assumption that energy requirements for differentiated cells are reduced compared to growing cells, being mostly post-mitotic and blocked in G0 phase. This cellular model was then used to test new Maytansinol-derivatives, synthesized within the TubInTrain consortium, as novel tubulin binders. Despite been proven as weak inhibitors of microtubules polymerization, one compound resulted effective in microtubules homeostasis modifiers, promoting microtubules dynamic properties. In addition, downstream 7 metabolomic analysis suggested that the compound could result in changes on cell metabolism. The results point towards impaired mitochondrial metabolism and neurotransmitter homeostasis, probably due to the alteration of the microtubule-mediated mitochondria trafficking and synaptic vesicles axonal transport. PC12 cells were transfected to overexpress either wild type or disease-associated A53T α-synuclein, and both microtubular compartment and cell metabolism were studied. We observed that wild type α-synuclein overexpression, but not disease-related A53T mutated α- synuclein, resulted in the decrease of acetylated-tubulin, suggesting more dynamic microtubules, enforcing the hypothesis of a putative interplay between tubulin acetylation and α-synuclein. In addition, our results show that modulating tubulin acetylation influences α- synuclein aggregation, supporting the idea that an impaired homeostasis of microtubules PTMs may be involved in the early stages of neurodegenerative processes. Regarding cell metabolism, wt α-synuclein overexpression seems not to significantly affect metabolic processes, while A53T-transfected cells were characterized by an imbalance of some amino acids, mostly related to oxidative stress response, pro-inflammatory environment and mitochondrial damage. These results indicate that the disease-associated A53T mutation of α-synuclein may exert its pathological properties directly binding to mitochondrial membranes and impairing their physiological functions. Finally, as the aggregation of α-synuclein is critically involved in the onset and progression of PD and related synucleinopathies, we employed a cell model that naturally showed increased oligomeric pathology due to α-synuclein overexpression, namely SK-N-SH cells, to study the protective impact of Oleuropein aglycone on α-synuclein aggregation and toxicity. For the detection of synuclein oligomers we took advantage of Proximity Ligation Assay technique, and according to our results, OA showed great potential in reducing the oligomeric burden in a neuronal cell model in a dose dependent manner. In addition, OA were proved capable of reducing the toxicity exerted by in vitro pre-formed α-synuclein fibrils. Our results were integrated with in vivo investigations in a C. elegans models of PD, suggesting that OA may effectively reduce the extent of different types of α-synuclein aggregates in both cellular and animal models of PD. This study offers a comprehensive view of Parkinson’s disease by examining the interactions between α-synuclein, mitochondrial function, microtubules, and metabolism in both cellular and in vivo models. It confirms the usefulness of PC12-based models and suggests potential therapeutic strategies, includingmicrotubule-targeting drugs and oleanolic acid. These findings enhance our understanding of PD and point toward new directions for future treatments.