Anti-oxidants in Parkinson's disease therapy: a critical point of view

Parkinson's disease (PD) is a degenerative neurological syndrome, which is characterized by the preferential death of dopaminergic (DAergic) neurons in the SubstantiaNigra. The pathogenesis of this disorder remains poorly understood and PD is still incurable. Currentdrug treatmentsare aimedprimarily for the treatmentof symptoms to improve the quality of life. Therefore, there is a need to find out new therapeutic strategies that not only provide symptomatic relief but also halt or reverse the neuronal damage hampering PD progression. Oxidative stress has been identified as one of the major contributors for the nigral loss in both sporadic and genetic forms of PD. In this review we first evaluate the current literature that link oxidative stress and mitochondrial dysfunction to PD. We then consider the results obtained through the treatmentof animal modelsor PD patients withmolecules that prevent oxidative stress or reduce mitochondrial dysfunction

Superoxide radical dismutation as protective mechanism to hamper the progression of Parkinson's disease

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

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field

Superoxide Dismutase (SOD)-mimetic M40403 is protective in cell and fly models of paraquat toxicity: Implications for Parkinson disease

Parkinson disease is a debilitating and incurable neurodegenerative disorder affecting 3c1-2% of people over 65 years of age. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. In this work, we demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, we then evaluated the effects using Drosophila melanogaster as an in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, our data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease

Leucine-rich repeat kinase 2 positively regulates inflammation and down-regulates NF-κB p50 signaling in cultured microglia cells

7openopenRusso, Isabella; Berti, Giulia; Plotegher, Nicoletta; Bernardo, Greta; Filograna, Roberta; Bubacco, Luigi; Greggio, Elisa*Russo, Isabella; Berti, Giulia; Plotegher, Nicoletta; Bernardo, Greta; Filograna, Roberta; Bubacco, Luigi; Greggio, Elis

Superoxide dismutating molecules rescue the toxic effects of PINK1 and parkin loss.

Reactive oxygen species exert important functions in regulating several cellular signalling pathways. However, an excessive accumulation of reactive oxygen species can perturb the redox homeostasis leading to oxidative stress, a condition which has been associated to many neurodegenerative disorders. Accordingly, alterations in the redox state of cells and mitochondrial homeostasis are established hallmarks in both familial and sporadic Parkinson's disease cases. PINK1 and Parkin are two genes which account for a large fraction of autosomal recessive early-onset forms of Parkinson's disease and are now firmly associated to both mitochondria and redox homeostasis. In this study we explored the hypothesis that superoxide anions participate in the generation of the Parkin and PINK1 associated phenotypic effect by testing the capacity of endogenous and exogenous superoxide dismutating molecules to rescue the toxic effects induced by loss of PINK1 or Parkin, in both cellular and fly models. Our results demonstrate the positive effect of an increased level of superoxide dismutase proteins on the pathological phenotypes, both in vitro and in vivo. A more pronounced effectiveness for mitochondrial SOD2 activity points to the superoxide radicals generated in the mitochondrial matrix as the prime suspect in the definition of the observed phenotypes. Moreover, we also demonstrate the efficacy of a SOD-mimetic compound, M40403, to partially ameliorate PINK1/Parkin phenotypes in vitro and in vivo. These results support the further exploration of SOD-mimetic compounds as a therapeutic strategy against Parkinson's disease

Mitochondrial dysfunction in adult midbrain dopamine neurons triggers an early immune response

Dopamine (DA) neurons of the midbrain are at risk to become affected by mitochondrial damage over time and mitochondrial defects have been frequently reported in Parkinson\u27s disease (PD) patients. However, the causal contribution of adult-onset mitochondrial dysfunction to PD remains uncertain. Here, we developed a mouse model lacking Mitofusin 2 (MFN2), a key regulator of mitochondrial network homeostasis, in adult midbrain DA neurons. The knockout mice develop severe and progressive DA neuron-specific mitochondrial dysfunction resulting in neurodegeneration and parkinsonism. To gain further insights into pathophysiological events, we performed transcriptomic analyses of isolated DA neurons and found that mitochondrial dysfunction triggers an early onset immune response, which precedes mitochondrial swelling, mtDNA depletion, respiratory chain deficiency and cell death. Our experiments show that the immune response is an early pathological event when mitochondrial dysfunction is induced in adult midbrain DA neurons and that neuronal death may be promoted non-cell autonomously by the cross-talk and activation of surrounding glial cells

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III 2, and IV (CI, CIII 2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII 2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology