Current Insights on Neurodegeneration by the Italian Proteomics Community

The growing number of patients affected by neurodegenerative disorders represents a huge problem for healthcare systems, human society, and economics. In this context, omics strategies are crucial for the identification of molecular factors involved in disease pathobiology, and for the discovery of biomarkers that allow early diagnosis, patients’ stratification, and treatment response prediction. The integration of different omics data is a required step towards the goal of personalized medicine. The Italian proteomics community is actively developing and applying proteomics approaches to the study of neurodegenerative disorders; moreover, it is leading the mitochondria-focused initiative of the Human Proteome Project, which is particularly important given the central role of mitochondrial impairment in neurodegeneration. Here, we describe how Italian research groups in proteomics have contributed to the knowledge of many neurodegenerative diseases, through the elucidation of the pathobiology of these disorders, and through the discovery of disease biomarkers. In particular, we focus on the central role of post-translational modifications analysis, the implementation of network-based approaches in functional proteomics, the integration of different omics in a systems biology view, and the development of novel platforms for biomarker discovery for the high-throughput quantification of thousands of proteins at a time

Mitochondrial alterations in Parkinson's disease human samples and cellular models

Abstract Mitochondrial impairment is one of the most important hallmarks of Parkinson's disease (PD) pathogenesis. In this work, we wanted to verify the molecular basis of altered mitochondrial dynamics and disposal in Substantia nigra specimens of sporadic PD patients, by the comparison with two cellular models of PD. Indeed, SH-SY5Y cells were treated with either dopamine or 1-methyl-4-phenylpyridinium (MPP + ) in order to highlight the effect of altered dopamine homeostasis and of complex I inhibition, respectively. As a result, we found that fusion impairment of the inner mitochondrial membrane is a common feature of both PD human samples and cellular models. However, the effects of dopamine and MPP + treatments resulted to be different in terms of the mitochondrial damage induced. Opposite changes in the levels of two mitochondrial protein markers (voltage-dependent anion channels (VDACs) and cytochrome c oxidase subunit 5β (COX5β)) were observed. In this case, dopamine treatment better recapitulated the molecular picture of patients' samples. Moreover, the accumulation of PTEN-induced putative kinase 1 (PINK1), a mitophagy marker, was not observed in both PD patients samples and cellular models. Eventually, in transmission electron microscopy images, small electron dense deposits were observed in mitochondria of PD subjects, which are uniquely reproduced in dopamine-treated cells. In conclusion, our study suggests that the mitochondrial molecular landscape of Substantia nigra specimens of PD patients can be mirrored by the impaired dopamine homeostasis cellular model, thus supporting the hypothesis that alterations in this process could be a crucial pathogenetic event in PD

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

The endogenous HBZ interactome in ATL leukemic cells reveals an unprecedented complexity of host interacting partners involved in RNA splicing

Adult T-cell leukemia/lymphoma (ATL) is a T-cell lymphoproliferative neoplasm caused by the human T-cell leukemia virus type 1 (HTLV-1). Two viral proteins, Tax-1 and HBZ play important roles in HTLV-1 infectivity and in HTLV-1-associated pathologies by altering key pathways of cell homeostasis. However, the molecular mechanisms through which the two viral proteins, particularly HBZ, induce and/or sustain the oncogenic process are still largely elusive. Previous results suggested that HBZ interaction with nuclear factors may alter cell cycle and cell proliferation. To have a more complete picture of the HBZ interactions, we investigated in detail the endogenous HBZ interactome in leukemic cells by immunoprecipitating the HBZ-interacting complexes of ATL-2 leukemic cells, followed by tandem mass spectrometry analyses. RNA seq analysis was performed to decipher the differential gene expression and splicing modifications related to HTLV-1. Here we compared ATL-2 with MOLT-4, a non HTLV-1 derived leukemic T cell line and further compared with HBZ-induced modifications in an isogenic system composed by Jurkat T cells and stably HBZ transfected Jurkat derivatives. The endogenous HBZ interactome of ATL-2 cells identified 249 interactors covering three main clusters corresponding to protein families mainly involved in mRNA splicing, nonsense-mediated RNA decay (NMD) and JAK-STAT signaling pathway. Here we analyzed in detail the cluster involved in RNA splicing. RNAseq analysis showed that HBZ specifically altered the transcription of many genes, including crucial oncogenes, by affecting different splicing events. Consistently, the two RNA helicases, members of the RNA splicing family, DDX5 and its paralog DDX17, recently shown to be involved in alternative splicing of cellular genes after NF-κB activation by HTLV-1 Tax-1, interacted and partially co-localized with HBZ. For the first time, a complete picture of the endogenous HBZ interactome was elucidated. The wide interaction of HBZ with molecules involved in RNA splicing and the subsequent transcriptome alteration strongly suggests an unprecedented complex role of the viral oncogene in the establishment of the leukemic state

verification of a parkinson s disease protein signature in t lymphocytes by multiple reaction monitoring

Diagnosis of Parkinson's disease, the second most common neurodegenerative disease, is based on the appearance of motor symptoms. A panel of protein biomarkers in the T-lymphocyte proteome was previously proposed as a Parkinson's disease signature. Here, we designed an LC–MS based method to quantitatively evaluate this protein signature by multiple reaction monitoring (MRM) in T-lymphocytes and peripheral blood mononuclear cells from a new cohort of nine patients with Parkinson's disease and nine unaffected subjects. Patients were classified using the discriminant function obtained from two-dimensional electrophoresis and protein amounts measured by MRM, thus assigning seven controls out of nine as true negatives and nine patients out of nine as true positives. A good discriminant power was obtained by selecting a subset of peptides from the protein signature, with an area under the receiver operating characteristic curve of 0.877. A similar result is achieved by evaluating all peptides of a selected pane..

The proteomic approach to investigate Parkinson's desease: pathogenetic mechanisms and biomarkers discovery.

The overall goals of basic research in improving the clinical approach to a disease are, on the one hand, to unravel its natural history, finding its cause and developing better treatments and, on the other hand, to find biomarkers that would permit to ameliorate the diagnosis, to distinguish among patient subtypes and to set up clinical trials of new therapeutic agents. The common threads of the two projects developed in this thesis are the technique used, proteomics, and the main object, Parkinson‟s disease (PD). Proteomics is a powerful methodology to investigate how protein expression is affected in the pathogenesis of a disease process and should be exploited both to investigate PD pathogenetic mechanisms and to discover peripheral biomarkers. In the first part of this thesis the project “A SH-SY5Y model to investigate PD pathogenetic mechanisms” is discussed. We investigated the expression pattern of cellular proteins following dopamine exposure in catecholaminergic SH-SY5Y human neuroblastoma cells overexpressing α-synuclein by a proteomic approach. Using 2D gel image analysis and ANOVA statistics we identified proteins that correlate with the experimental conditions in our model. Significant changes in specific cellular processes were observed, such as cytoskeleton structure and regulation, mitochondrial function, energetic metabolism, protein synthesis and neuronal plasticity. Proteins were analyzed by a network enrichment tool that automatically enriched the network model in terms of the most relevant proteins that seem to be missing. Moreover, the bioinformatic approach found out the enriched Gene Ontology (GO) categories, allowing us to draw attention to the NF-κB pathway. To validate experimentally the hypothesis of the NF-κB involvement, we performed a luciferase gene reporter assay that revealed a downregulation of the transcription factor activation by α-synuclein increased expression and its complete quenching by dopamine exposure. Each element arising from the present study could represent a valuable starting point for focused investigations aimed to better understand key issue of PD pathogenesis. For instance, a protein that completely disappeared after dopamine treatment was VDAC2, a porin of the outer mitochondrial membrane. Since the impairment of mitochondrial function is considered among the major pathogenetic factors of neurodegeneration in PD, we further investigated the regulation of VDAC2 degradation and the role of its phosphorylation. Accumulating evidence supports a specific role of DJ-1 in protecting dopaminergic neurons from dopamine itself. Since this protein act as a pool of different forms possibly associated to different function and localization, we analyzed DJ-1 2D electrophoresis pattern in SH-SY5Y cells after exposure to dopamine; we observed a specific increase in the most acidic forms in the pattern together with a significant decrease of the most basic spot. Unlike cells exposed to generic oxidative conditions, no additional shift was observed. The results are corroborated by a meta-analysis of the literature showing that in the absence of dopamine treatment the specific acidic form is underrepresented. The second part of the thesis is focused on the project “Dopamine response of dopaminergic circulating cells: the Jurkat cell model”. The functions of immune cells are regulated not only by cytokines, but also by several neurotransmitters. In particular, peripheral blood lymphocytes possess a complex dopaminergic regulatory system. Human T-lymphocytes express all five dopamine receptors (D1-D5), each of which exerts different actions on the regulation of T-cell functions. The Jurkat human leukemia CD4+ T-cell line has been thoroughly used and characterized as a suitable cell model to investigate T-cell signaling and apoptosis, nevertheless its characterization as a model of circulating dopaminergic cells was never reported before. In this regard, we characterized the dopaminergic system in Jurkat cells and through a proteomic approach we analyzed their response to a dopamine challenge in order to highlight metabolic pathways that could specifically reveal alterations linked to PD. A dopamine challenge, with no effect on cell viability (50 μM), induced quantitative changes in the protein expression pattern. The proteins that showed quantitative differences were identified by peptide mass fingerprinting and analyzed in terms of both interaction network and GO classification enrichment. We obtained a significant model for network enrichment and a functional association to GO classification (unfolded protein binding). Eventually, we observed a modification of β-actin and 14-3-3 two-dimensional pattern following dopamine exposure. In both projects, by combining the experimental and the bioinformatic approach, we fulfilled the expectations for proteomics to generate new hypotheses. Of course, one must consider that models have limitations and that hypotheses need further validation, but they could be a good starting-point to unravel biochemical pathways altered in PD, a disease with a poorly understood etiology

Proteomics in Parkinson's disease: An unbiased approach towards peripheral biomarkers and new therapies

Parkinson's disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's disease is a socio-economic trouble and a major challenge for the public health system. Parkinson's disease treatment is merely symptomatic, as clinical symptoms appear when about 70\% of the involved neurons are lost and potential disease-modifying/neuroprotective therapies would have no effect. In turn, the availability of an objective measure that allows early diagnosis would strongly impact on the costs that biotech- and pharma-companies will sustain in order to develop disease-modifying therapies. The establishment of suitable models to investigate the mechanisms of Parkinson's disease progression and, on the other hand, the discovery and validation of selective and specific molecular biomarkers for early and differential diagnosis are indeed two important goals for a better management of the disease. In this review, we focus on cellular and animal models of Parkinson's disease by describing their advantages and limitations as useful tools to identify pathogenetic pathways that deserve further exploitation. In parallel, we discuss how proteomics may provide a potent tool to observe altered pathways in models or altered biomarkers in patients with an unbiased, hypothesis-free approach.Parkinson's disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's disease is a socio-economic trouble and a major challenge for the public health system. Parkinson's disease treatment is merely symptomatic, as clinical symptoms appear when about 70% of the involved neurons are lost and potential disease-modifying/neuroprotective therapies would have no effect. In turn, the availability of an objective measure that allows early diagnosis would strongly impact on the costs that biotech- and pharma-companies will sustain in order to develop disease-modifying therapies. The establishment of suitable models to investigate the mechanisms of Parkinson's disease progression and, on the other hand, the discovery and validation of selective and specific molecular biomarkers for early and differential diagnosis are indeed two important goals for a better management of the disease. In this review, we focus on cellular and animal models of Parkinson's disease by describing their advantages and limitations as useful tools to identify pathogenetic pathways that deserve further exploitation. In parallel, we discuss how proteomics may provide a potent tool to observe altered pathways in models or altered biomarkers in patients with an unbiased, hypothesis-free approach. © 2011 Elsevier B.V.