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Cytotoxic effects of 6-OHDA towards dopaminergic neuron-like cells are independent of LRRK2 activation

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Beatriz Pelegrini Bosque
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
Matheus de Castro Fonseca; Mirian Akemi Furuie Hayashi; Tatiana Carla Tomiosso
Advisor: Matheus de Castro Fonseca

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, generating progressive debilitations for the patient, such as motor impairment, cognitive and psychiatric deficits. About 10% of reported cases of the disease are due to genetic inheritance, also called familial PD, while 90% have unknown causes, i.e., idiopathic (iDP). Studies suggest that mutations in ?20 genes cause rare genetic parkinsonism, including the gene encoding leucine-rich protein kinase 2 (LRRK2). To date, all pathogenic mutations in this gene result in hyperactivation of LRRK2 protein kinase activity. Interestingly, this exacerbated function has also been described in PD patients who did not carry mutations, suggesting that it may be a risk factor for the development of the sporadic form of the disease. Thus, we propose to evaluate possible changes in subcellular localization, expression and phosphorylation of wild-type LRRK2 using a pharmacological model of PD based on 6-hydroxydopamine (6-OHDA) administration in vitro. Initially, human neuroblastoma SH-SY5Y cells were cultured in DMEM F/12 medium supplemented with 2% SFB, 1% PSA and 10?M retinoic acid for 10 days in order to induce a dopaminergic neuron-like phenotype. Once differentiation was completed, these cells were treated with 6-OHDA 100?M for 1hr, 4hs or 24hs and then subjected to cytosolic and mitochondrial calcium (Ca2+) signaling, reactive oxygen species (ROS) generation and cell viability (MTS) assays, as well as protein expression/phosphorylation evaluation by western blot and by organotypic culture experiments. Differentiation was proven by analysis of cell morphology and expression of specific neuronal markers. Differentiated cells developed extensive neurites and stellate morphology, and expressed more neuronal markers when compared to non-differentiated cells. Assays of cytosolic Ca2+ signaling demonstrated that treatments with 6-OHDA generated a diminished response as well as a failure to normalize basal Ca2+ levels after stimulation, which was also found for Ca2+ signaling in mitochondria, indicating that this organelle is highly affected. It was also observed that 6-OHDA caused an increase in the amount of intracellular ROS, which may be the cause of mitochondrial impairment, and caused a reduction in cell viability levels relative to controls. When analyzing the effects of 6-OHDA on LRRK2, it was observed that 6-OHDA did not alter the expression or phosphorylation of the protein in dopaminergic neurons or in organotypic cultures at any of the treatment times. In addition, two known targets activated by LRRK2 (a-syn and Rab8a) were also unchanged in expression and phosphorylation. Thus, we concluded that the cytotoxic effects of 6-OHDA on dopaminergic neurons are independent of LRRK2 protein activation, thus involving other cellular mechanisms (AU)

FAPESP's process: 19/24511-0 - The role of LRRK2 and Rab GTPases in the neurodegeneration during sporadic Parkinson's Disease
Grantee:Beatriz Pelegrini Bosque
Support Opportunities: Scholarships in Brazil - Master