Parkinson's disease plasma biomarkers: An automated literature analysis followed by experimental validation

Diagnosis of Parkinson's disease (PD) is currently assessed by the clinical evaluation of extrapyramidal signs. The identification of specific biomarkers would be advisable, however most studies stop at the discovery phase, with no biomarkers reaching clinical exploitation. To this purpose, we developed an automated literature analysis procedure to retrieve all the background knowledge available in public databases. The bioinformatic platform allowed us to analyze more than 51,000 scientific papers dealing with PD, containing information on 4121 proteins. Out of these, we could track back 35 PD-related proteins as present in at least two published 2-DE maps of human plasma. Then, 9 different proteins (haptoglobin, transthyretin, apolipoprotein A-1, serum amyloid P component, apolipoprotein E, complement factor H, fibrinogen γ, thrombin, complement C3) split into 32 spots were identified as a potential diagnostic pattern. Eventually, we compared the collected literature data to experimental gels from 90 subjects (45 PD patients, 45 non-neurodegenerative control subjects) to experimentally verify their potential as plasma biomarkers of PD

Impact of cysteine 106 oxidation on function and localisation of the Parkinson’s disease-related protein DJ-1

Postponed access: the file will be accessible after 2020-06-11Masteroppgave i molekylærbiologiMOL399MAMN-MO

Proteomics and network analysis identify common and specific pathways of neurodegeneration

Neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) are multi-factorial in nature, involving several genetic mutations (in coding or regulatory regions) and epigenetic and environmental factors. The main clinical manifestation (movement disorders, cognitive impairment and/or psychiatric disturbances) depends on the neuron population being primarily affected. Complex and multifactorial neurodegenerative diseases can be investigated using a holistic approach that can give a global view about the pathogenetic process and shed light on specific and generic pathways of neurodegeneration. Proteomics offers a global molecular snapshot of proteins and consequently of processes that may influence neuronal death. The proteome in fact provides a dynamic view of what is happening in the system under investigation, because the expression of proteins, their abundance, their localization in tissues or cells, the type and amount of their post-translational changes depend from the environment and from the cellular physiological state. Therefore, all the projects presented in this thesis, by combining bioinformatics tools with proteomics, aimed at highlighting biochemical processes shared by different neurodegenerative diseases and diseasespecific pathways, which may justify the degeneration of dopaminergic neurons in PD. Finally, a focus on the mitochondrial interactome and proteome intended to elucidate important specific steps of the degenerative process in PD

Parkinson's disease biomarkers in peripheral blood T lymphocytes: identification through a proteomic approach

All the spots of the models or showing correlation with one of the mentioned parameters were excised from the gels and identified by LC-MS/MS fragmentation: some of the most important and recurring identification were beta fibrinogen and transaldolase; in particular, 4 spots correspond to beta fibrinogen and 2 spots correspond to transaldolase; all together, these 6 spots contribute to the prediction model in a very remarkable way. For this reason, we validated by Western blot total beta fibrinogen and transaldolase changes to verify their proper identification and confirm the differences observed by 2-DE with another technique (Western blotting). Worth of note, the level of some proteins was modified by levodopa and dopamine agonists therapy in T lymphocytes: this is a demonstration that these cells are sensitive to long term dopaminergic stimulation and that they are a valid tool to investigate dopaminergic imbalance at a peripheral level. Additional experiments were conducted in order to deepen the role of fibrinogen in T cells, and the reason why its volume is reduced in PD patients: this study is currently going on, but preliminary results indicate that it is internalized by T cells from plasma. In conclusion, in this thesis T lymphocyte proteome changes have been shown to be a valid tool to recognize and classify PD patients. It has been proposed a model using a rationale of similarity between affected cells (dopaminergic neurons) and probed system (T lymphocytes): we expect that our system will reasonably provide information on the effectiveness of future (or under development) drugs designed to interfere with specific cell death mechanisms targeting nigral neurons. Moreover, our system possess most features required for candidate PD biomarkers (specificity, indication of progression, accessibility). Next step of this research is leading the current finding to the clinical practice. This objective will be reached only through large collaborative networks, that will allow us to select large cohorts of subjects with high heterogeneity in terms of gender, age, ethnicity, clinical phenotype; the use of a different and less time consuming technique in the clinical phase is advisable

Parkinson's disease biomarkers in peripheral blood T lymphocytes: identification through a proteomic approach

All the spots of the models or showing correlation with one of the mentioned parameters were excised from the gels and identified by LC-MS/MS fragmentation: some of the most important and recurring identification were beta fibrinogen and transaldolase; in particular, 4 spots correspond to beta fibrinogen and 2 spots correspond to transaldolase; all together, these 6 spots contribute to the prediction model in a very remarkable way. For this reason, we validated by Western blot total beta fibrinogen and transaldolase changes to verify their proper identification and confirm the differences observed by 2-DE with another technique (Western blotting). Worth of note, the level of some proteins was modified by levodopa and dopamine agonists therapy in T lymphocytes: this is a demonstration that these cells are sensitive to long term dopaminergic stimulation and that they are a valid tool to investigate dopaminergic imbalance at a peripheral level. Additional experiments were conducted in order to deepen the role of fibrinogen in T cells, and the reason why its volume is reduced in PD patients: this study is currently going on, but preliminary results indicate that it is internalized by T cells from plasma. In conclusion, in this thesis T lymphocyte proteome changes have been shown to be a valid tool to recognize and classify PD patients. It has been proposed a model using a rationale of similarity between affected cells (dopaminergic neurons) and probed system (T lymphocytes): we expect that our system will reasonably provide information on the effectiveness of future (or under development) drugs designed to interfere with specific cell death mechanisms targeting nigral neurons. Moreover, our system possess most features required for candidate PD biomarkers (specificity, indication of progression, accessibility). Next step of this research is leading the current finding to the clinical practice. This objective will be reached only through large collaborative networks, that will allow us to select large cohorts of subjects with high heterogeneity in terms of gender, age, ethnicity, clinical phenotype; the use of a different and less time consuming technique in the clinical phase is advisable

Proteome analysis of human substantia nigra in Parkinson's disease

<p>Abstract</p> <p>Background</p> <p>Parkinson's disease (PD) is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra.</p> <p>Results</p> <p>The gross number of differentially regulated proteins in PD was 221. In total, we identified 37 proteins, of which 16 were differentially expressed. Identified differential proteins comprised elements of iron metabolism (H-ferritin) and glutathione-related redox metabolism (GST M3, GST P1, GST O1), including novel redox proteins (SH3BGRL). Additionally, many glial or related proteins were found to be differentially regulated in PD (GFAP, GMFB, galectin-1, sorcin), as well as proteins belonging to metabolic pathways sparsely described in PD, such as adenosyl homocysteinase (methylation), aldehyde dehydrogenase 1 and cellular retinol-binding protein 1 (aldehyde metabolism). Further differentially regulated proteins included annexin V, beta-tubulin cofactor A, coactosin-like protein and V-type ATPase subunit 1. Proteins that were similarly expressed in healthy or diseased substantia nigra comprised housekeeping proteins such as COX5A, Rho GDI alpha, actin gamma 1, creatin-kinase B, lactate dehydrogenase B, disulfide isomerase ER-60, Rab GDI beta, methyl glyoxalase 1 (AGE metabolism) and glutamine synthetase. Interestingly, also DJ-1 and UCH-L1 were expressed similarly. Furthermore, proteins believed to serve as internal standards were found to be expressed in a constant manner, such as 14-3-3 epsilon and hCRMP-2, thus lending further validity to our results.</p> <p>Conclusion</p> <p>Using an approach encompassing high sensitivity and high resolution, we show that alterations of SN in PD include many more proteins than previously thought. The results point towards a heterogeneous aetiopathogenesis of the disease, including alterations of GSH-related proteins as well as alterations of proteins involved in retinoid metabolism, and they indicate that proteins involved in familial PD may not be differentially regulated in idiopathic Parkinson's disease.</p

Pathophysiological mechanisms in Parkinson`s Disease and Dystonia – converging aetiologies

In this thesis I used a range of experimental approaches including genetics, enzyme activity measurements, histology and imaging to explore converging pathophysiological mechanisms of Parkinson`s disease and dystonia, two conditions with frequent clinical overlap. First, based on a combined retro- and prospective cohort of patients, using a combination of lysosomal enzyme activity measurements in peripheral blood and brain samples, as well as a target gene approach, I provide first evidence of reduced levels of enzyme activity in Glucocerebrosidase and the presence of GBA mutations, indicating lysosomal abnormality, in a relevant proportion of patients with dystonia of previously unknown origin. Second, based on a retrospective cohort of patients, I detail that a relevant proportion of genetically confirmed mitochondrial disease patients present with a movement disorder phenotype - predominantly dystonia and parkinsonism. Analysing volumetric MRI data, I describe a patterned cerebellar atrophy in these particular patients. This also includes the first cases of isolated dystonia due to mitochondrial disease, adding the latter as a potential aetiology for dystonia of unknown origin. Third, I used a combination of post-GWAS population genetic approaches and tissue-based experiments to explore in how far the strong association between advancing age and Parkinson ́s disease is mediated via telomere length. Although the initial finding of an association between genetically determined telomere length and PD risk did not replicate in independent cohorts, I provide evidence that telomere length in human putamen physiologically shortens with advancing age and 3 is regulated differently than in other brain regions. This is unique in the human brain, implying a particular age-related vulnerability of the striatum, part of the nigro-striatal network, crucially involved in PD pathophysiology. I conclude by discussing the above findings in light of the current literature, expand on their relevance and possible direction of future experiments

Dopamine and dopamine-quinones toxicity in Parkinson's disease: cellular models revealing a possible role for superoxide dismutases

Parkinson’s disease is a widespread neurodegenerative disorder that affect 2% of the population above the age of 60. The hallmark of the pathology is the preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta of the midbrain, and the presence of proteinaceous inclusions called Lewy bodies in the surviving neurons (Braak 2004). In 10% of the cases, the disease is linked to mutation on several genes, among them α-synuclein, DJ-1, PARKIN, PINK1 and LRRK2, but in the vast majority of the cases the aetiology is still unknown (sporadic PD) (Gwinn-Hardy 2002). Post mortem studies and in vitro and in vivo PD model have revealed a possible interconnection between genetic and sporadic PD, which involves both mitochondrial dysfunction and oxidative stress as central players in the pathogenesis of the disease (Gilgun-Sherki Y. et al. 2001, Mythri R. B. et al. 2011). Oxidative stress is a condition characterized by the inability of the cellular antioxidant defences to cope with the production of reactive oxygen species (ROS). This condition of unbalance between the production and the clearance of ROS causes irreversible damage to cellular components such as lipids, proteins and DNA, leading eventually to cell death (Lotharius et al. 2002). Among the enzymes implicated in the detoxification of ROS, are superoxide dismutases (SODs) that catalyze the dismutation of superoxide anion into molecular oxygen and hydrogen peroxide (Fridovich 1995). Since oxidative stress does not explain alone the selectivity death of dopaminergic neurons, the main working hypothesis is that dopamine itself could have a central role. Under physiological conditions, dopamine is synthesized in the cytosol and stored in synaptic vesicles by the action of Vesicular Monoamine Transporter (VMAT2) where it is stabilized by the low pH (Erickson, J. D 1992). If the amount of cytosolic DA exceeds the physiological concentration, DA is metabolized to the non-toxic metabolite 3,4-dihydroxyphenylacetic acid and hydrogen peroxide by the action of monoamine oxidase (MAO) and aldehyde dehydrogenase, or sequestered into lysosomes where it can auto-oxidize to form neuromelanin (NM). If not buffered by these pathways, cytosolic DA can be oxidized to DA-quinone (DAQs) (spontaneously or enzymatically), (Sulzer, D., 2000, Elsworth, J. D. 1997), a reaction that also leads to the formation of ROS. On these premise, we evaluated two line of research using a cellular model for PD (SH SY5Y cell line): one concerning about the effect of dopamine and its oxidized forms on cellular viability, the second one on the potential role of superoxide dismutases (1 and 2) over expression. From the use of different techniques we started to evaluate which kind of cell death pathway was activated by dopamine and DAQs. Looking for the presence of nuclear fragmentation, that is one of the later stages of apoptosis, we determined that both dopamine and DAQs induce cell death via apoptosis but the dopamine toxicity depends on its internalization by the action of the dopamine transporter (DAT), since the pre-treatment of cells with GBR 12909 (a DAT inhibitor) had a rescue effect. To confirm the apoptotic pathway we also evaluated another hallmark of apoptosis (one of the former stages of the apoptotic cascade): phosphatidil-serine externalization (PS) using ANNEXIN-V-FLUOS; a specific probe for PS. Using flow cytometry we confirm that both dopamine and DAQs induce cell death via apoptosis. Next we wanted to evaluate if dopamine and DAQs exert their toxicity from extracellular environment or they are required to enter in the cells. Treating cells with GBR12909, we demonstrate that dopamine needs to enter cells to exert its toxicity (since the treatment with the DAT inhibitor rescues cells from DA toxicity) while DAQs toxicity was not affected by this treatment leading to cell death. Since oxidative stress is one of the mechanisms that have been implicated in the pathogenesis of PD, and the chemistry of dopamine (auto-oxidation and enzyme-mediated oxidation) leads to the production of ROS, we evaluate the production of mitochondrial superoxide anion using a specific probe. The data demonstrate that only the auto oxidation of dopamine leads to the production of superoxide anion and dopamine is required to enter cell to exert its effect. To dissect more in depth the toxicity mechanism of both dopamine and DAQs, and since only cytosolic dopamine led to the production of mitochondrial superoxide anion, we asked if this two different oxidation processes activated different cell death pathways (the major are the mitochondrial one and the one mediated by death receptor) or not. From preliminary data we observed a marked difference in the activation of caspase 3 and the subsequent cleavage and inactivation of Poly (ADP) ribose polymerase (PARP) due to DAQs treatment convincing us to proceed in the investigation of the possible differences between this different oxidation processes. The second part of the work was focused on the role for superoxide dismutases 1 and 2 against dopamine and DAQs cytotoxicity since previous data demonstrated a role in superoxide anion production and induction of cell death in the case of cytosolic dopamine. Over expression of both SOD1 and SOD2 revealed a protective effect against dopamine cytotoxity, while they were not able to counteract DAQs-induced cell death. In the present work the main working hypothesis was that oxidative stress induced by dopamine and its oxidized forms accumulation could have a central role in the specific dopaminergic cell loss in Parkinson’s disease. The data obtained so far seems to highlights that dopamine and DAQs activates different apoptotic pathway that are superoxide anion-dependent for DA and superoxide anion-independent for DAQs. Since oxidative stress is considered one of the mechanism that interconnect genetic form and sporadic forms of the pathology and dopamine, in its oxidative chemistry, leads to the production of ROS, understanding which cell death pathways are activated and to which extent, is crucial to develop a therapy to counteract the start and the progression of the pathology. Data from the over expression of SODs demonstrate that compounds that counteract the production of superoxide anion (like SOD-mimetics that are currently used for other diseases) could have a protective role against the oxidative stress and the subsequent cell death condition induced by dopamine. Also compounds that block the activation of the apoptotic cascade induced by dopamine and DAQs could rescue cells from dying in this neurodegenerative diseas