THE ROLE OF PARKIN IN MODULATING MT SYSTEM IN GENE-BASED EXPERIMENTAL MODELS OF PARKINSON'S DISEASE
Creators: name: DE GREGORIO, CARMELITA
Publisher: name: Universit� degli Studi di Milano
Year: 2016

description: The molecular mechanisms underlying the dopaminergic neuronal death in Parkinson's disease (PD) result still unclear and thus proven strategy to prevent the disease also remains unknown and an unmet priority. Several pathogenic pathways have been implicated in PD pathogenesis and, among them, microtubule (MT) dysfunction is emerging as a contributing factor of the disease. MT cytoskeleton is a complex network essential for neuronal morphogenesis and its proper regulation is crucial for neuronal survival and functions including axonal transport. MTs are common targets for several PD inducing toxins and PD-linked proteins including parkin that, beyond its E3 ubiquitin ligase activity, plays a role in regulating mitochondrial homeostasis and trafficking. Some data, derived from murine and human cell cultures, provide evidences that parkin mutations, which are responsible for both familial and sporadic forms of PD, affect MT system. In this scenario, we analyzed in detail the interplay between parkin, MT system and mitochondrial axonal transport using gene-based murine models of the disease. First, we investigated MT dysfunction in PARK2-Q311X transgenic mice, a model that resembles the hallmark characteristic of the disease in age dependent way. Our initial analyses showed that mice at 6 and 16 weeks of age did not show deficits in striatal innervations and depletion of tyrosine hydroxylases (TH), indicating that they could be suitable to study the early pathogenic phases that precede neuronal death. Thus, we studied the impact of parkin Q311X mutation on MT stability and mitochondrial homeostasis in 6 and 16 weeks old mice with the aim to pinpoint the involvement of their dysfunction as early event in neurodegeneration. We found that Q311X point mutation leads to the unbalance of post-translationally modified tubulins (PTMs), which are associated with differences in MT stability, inside dopaminergic neurons. On the contrary, none alteration was observed for mitochondrial dynamics and distribution in nigrostriatal fibers at these very early time points. Notably, we also reported that the unbalance of tubulin PTMs occurs in PARK2 knockout (KO) model during aging (2, 7 and 24 months of age) and precedes the block of mitochondrial transport within dopaminergic fibers. Collectively, our results from PARK2 mouse models support the concept that early ?-tubulin PTMs alterations might play an important role in PD etiopathogenesis. Next, we moved to primary neuronal cultures obtained from midbrain of mouse embryos as experimental model for investigating in detail the effect of parkin in modulating MT dynamics and mitochondria motility. Morphometric analyses, performed on primary neurons isolated from PARK2 KO and heterozygous mice showed that parkin deficiency affects axonal outgrowth. To test whether the impact of parkin deficiency on morphology is linked to MT dysfunction, we firstly investigated MT dynamics. Beyond the enrichment in the level of Tyr tubulin (marker of dynamic MTs), we observed that parkin deficiency increases MT dynamics by live cell imaging. Moreover, we assessed MT-based axonal transport and our results confirmed that loss of parkin affects mitochondria movement. We showed that this alteration depends on MT destabilization as it is rescued by MT stabilizer paclitaxel in primary neurons and also in PARK2- silenced PC12 cells. Overall, these data pinpoint parkin as a regulator of tubulin PTMs and MT dynamics in neurons and reinforce the idea that MT dysfunction may be crucial and represent an early step in the chain of events leading to dopaminergic neuron death.