Global protein turnover rates of primary Astrocytes in a mouse model of Parkinson's disease

Parkinson"s disease (PD) is a complex neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. One of its main pathological hallmarks is the presence of intracellular protein inclusions (Lewy Bodies) in neuronal and glial cells.

While several genes have been associated with PD etiology, point mutations in leucine-rich repeat kinase 2 (LRRK2)-encoding gene stand out as the most common cause of the familial form. LRKK2 is a multidomain kinase involved in many cellular processes, from vesicle trafficking to the regulation of lysosome homeostasis. All pathogenic mutations seem to directly increase its kinase activity, resulting in abnormal modulation of its substrates. However, how this impacts PD progression is still not completely understood. In addition, the role of LRRK2 in glial cells such as astrocytes and the involvement of these cells in the degeneration of neurons and in the neuroinflammation process have been underexplored, as most studies focused on neurons.

Since impaired protein homeostasis and accumulation of protein aggregates are key features of PD pathogenesis, we investigated the potential contribution of G2019S-LRRK2, the most frequently observed mutated form, on the global turnover of proteins in astrocytes at the early stages of the disease.

To systematically estimate protein turnover rates, we carried out a dynamic-SILAC experiment on primary astrocytes of wildtype and G2019S-LRRK2 knocked-in mice. A pre-treatment for 24 hours with pre-formed fibrils of alpha-synuclein, the main component of Lewy Bodies, was performed to evaluate possible differences in turnover rates in simulated pathological conditions. Cells were collected at three different time points (1 day, 4 days, 6 days) and proteins were analyzed by data-dependent acquisition (DDA) LC-MS/MS. The turnover rate was determined for each protein by applying a linear model at the peptide-level data.

Our results indicate that the G2019S mutation of LRRK2 deeply affects the half-lives of proteins involved in the ER-Golgi transport and cell adhesion. As expected, some of these proteins are known substrates of LRRK2. Interestingly, the treatment with alpha-synuclein does not appear to modify protein turnover rates of wildtype astrocytes significantly. In contrast, the same treatment in astrocytes carrying the G2019S-LRRK2 mutation strongly alters the half-life of proteins, in particular those belonging to the respiratory chain complexes and those that participate in vesicle-mediated transport. Moreover, the estimation of protein abundance levels suggests a possible inhibition of autophagy through the activation of mTORC1, as already observed for other PD models.