Abstract
Parkinson's disease (PD) is a degenerative neurological syndrome characterized by the preferential loss of dopaminergic (DAergic) neurons in the Substantia Nigra pars compacta. PD is still incurable and conventional therapies treat only symptoms to improve the quality of life. Therefore, there is a impelling need to find out new therapeutic strategies that not only provide symptomatic relief but also halt or reverse the neuronal damage hampering PD progression. Even though the pathogenesis of this disorder remains poorly understood, oxidative stress has been identified as one of the major contributors for the nigral loss in both sporadic and genetic forms of the disease. In particular, the selective vulnerability of DAergic neurons to oxidative stress might be ascribed to dopamine (DA) metabolism, which occurs in the cytosol and represents in itself a relevant pathway for superoxide radicals production. The main hypothesis of this thesis is that the inhibition of reactive oxygen species (ROS) overproduction might delay, block or prevent the degenerative process that occurs in PD patients. In this scenario, our project was addressed to study in vitro and in vivo the potential protective role of the superoxide dismutase (SOD) enzymes and SOD mimetic compounds against oxidative injury, related to PD, adopting two experimental paradigms. We focused on SODs because they exert a crucial function in cellular antioxidant defense, promoting the elimination of superoxide anion.
The first experimental paradigm was represented by the herbicide paraquat (PQ) whose mechanism of action relies on the production of oxidative stress and it is epidemiologically linked to sporadic PD. The second one, which has been used to model a familial form of PD, was based on PINK1 deficiency. Indeed, PINK1 gene mutations have been identified as cause of recessive early-onset parkinsonism. This gene encodes for a serine/threonine kinase that is involved in the mitochondrial quality control and in the regulation of cellular oxidative status.
To evaluate whether SODs might have a protective activity against PQ toxicity or PINK1 deficiency, the cytosolic and mitochondrial SODs, respectively SOD1 and SOD2, were overexpressed in the human neuroblastoma SH-SY5Y cells and in Drosophila melanogaster. In cells and flies, the overexpression of the mitochondrial isoform rescued acute PQ toxicity. The selective effect observed seems to be associated to an intrinsic mechanism of acute treatment, which strongly compromise mitochondria, increasing ROS in these organelles and promoting their fragmentation. On the contrary, in flies the cytosolic isoform ameliorated motor dysfunctions induced by a chronic PQ exposure, even when SOD1 was overexpressed exclusively into the DAergic neurons. These observations indicate that the cytosolic compartment is particularly affected by chronic PQ treatment suggesting that other oxidative processes in the cytosol of DAergic cells, such as DA metabolism, might amplify PQ-induced oxidative stress making them particularly vulnerable. In SH-SY5Y cells, PINK1 deficiency resulted in mitochondrial fragmentation. Even in this case, SODs appeared protective rescuing the phenotype. However, while SOD1 overexpression slightly reduced these mitochondrial alterations, SOD2 seemed to reverse mitochondrial fragmentation allowing the maintenance of a healthy mitochondrial network. In flies, loss of PINK1 induced a severe motor impairment, which was rescued only by the overexpression of the cytosolic isoform suggesting that the protein might be involved in other pathways that are not strictly correlated with mitochondrial functioning.
Once the beneficial activity of SODs has been demonstrated, we then investigated the therapeutic potential use of a SOD-mimetic compound, M40403. We found that the molecule was able to protect cells and flies against the oxidative damage induced by both acute and chronic PQ exposure. In addition, the SOD mimetic was effective also in PINK1 deficient cells and flies reducing, respectively, mitochondrial fragmentation and locomotor defects. Finally, M40403 administration in SOD1 and SOD2 deficient flies partially replaced the loss of both isoforms suggesting that it can act at cytosolic and mitochondrial level.
Overall, these findings demonstrate that specific SOD-mimetic compounds can be efficacious in reducing oxidative stress and should be further explored as therapeutic agents to hamper the progression of PD.
In parallel, we developed a second research line which was aimed to the characterization of two human neuroblastoma cell lines in order to identify, between them, the most reliable cellular model for PD studies.
Cellular models are largely used to study in vitro the molecular mechanisms underlying DAergic degeneration in PD. Although their use presents several advantages, cell lines do not always recapitulate morphological and neurochemical properties of DAergic neuronal cells. Considering the relevance of DA metabolism in the pathogenesis of PD, the DAergic phenotype is an important requirement. Human neuroblastoma cell lines are commonly used as models in PD research, although they are undifferentiated, do not exhibit markers of mature neurons and appear able to synthetize different neurotransmitter, in particular the catecholamines DA and noradrenaline (NA). For this reason, we studied the ability of three different agents, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) and staurosporine to drive neuronal differentiation toward a DAergic phenotype in SH-SY5Y and BE(2)-M17 cells. The first cell line is largely adopted and studied, even though the phenotype acquired upon differentiation is still a debated issue. In contrast, the second one is poorly characterized and might represent a valid alternative cellular system. In this thesis, we first investigated the acquisition of neuronal-like features in terms of growth inhibition, cell morphology and neuronal markers expression. Our results indicated that staurosporine and RA were the most efficient treatments to inhibit cell growth, respectively in SH-SY5Y and BE(2)-M17. Furthermore, in both cell lines, RA and staurosporine promoted the formation a complex network of neuritic extensions and the expression of mature neuronal markers. To evaluate whether the differentiation promotes a DAergic or NAergic phenotype in these cell lines, we analyzed the expression profile of the major genes involved in DA and NA metabolism and the intracellular content of these neurotransmitters. In SH-SY5Y cells, RA and TPA induced the down-regulation of DA- and NA-related genes as well as a decrease of neurotransmitter amounts compared to undifferentiated cells, indicating the loss of the catecholaminergic phenotype. On the contrary, staurosporine treatment resulted in the up-regulation of all these genes and an increase of NA content, enhancing the NAergic phenotype. Surprisingly, in BE(2)-M17, DA and NA levels detected in undifferentiated cells were considerably more elevated than in SH-SY5Y which suggests that these cells presents a more pronounced catecholaminergic phenotype. The latter was not affected by TPA and RA treatments, which did not substantially alter gene expression and the amount of neurotransmitters. In contrast, staurosporine promoted the up-regulation of the genes involved in metabolism of DA and NA and an increase of their intracellular amounts, indicating a relevant enhancement of the observed phenotype.
These results indicate that the BE(2)-M17 cell line emerges as a new experimental model with a catecholaminergic phenotype that differs substantially from those of SH-SY5Y cells, suggesting different fields of application for the two cell line
