The glycatome

Funding Information: This study was supported by iNOVA4Health UID/Multi/04462/2013 – UIDB/04462/2020 and UIDP/04462/2020 , a program financially supported by Fundação para a Ciência e a Tecnologia (FCT) / Ministério da Ciência, Tecnologia e Ensino Superior, through national funds; and by Sociedade Portuguesa de Diabetologia . AC was supported by PD/BD/136863/2018 , FCT.We are witnessing a considerable increase in the incidence of Parkinson's disease (PD), which may be due to the general ageing of the population. While there is a plethora of therapeutic strategies for this disease, they still fail to arrest disease progression as they do not target and prevent the neurodegenerative process. The identification of disease-causing mutations allowed researchers to better dissect the underlying causes of this disease, highlighting, for example, the pathogenic role of alpha-synuclein. However, most PD cases are sporadic, which is making it hard to unveil the major causative mechanisms of this disease. In the recent years, epidemiological evidence suggest that type-2 diabetes mellitus (T2DM) individuals have higher risk and worst outcomes of PD, allowing to raise the hypothesis that some dysregulated processes in T2DM may contribute or even trigger the neurodegenerative process in PD. One major consequence of T2DM is the unprogrammed reaction between sugars, increased in T2DM, and proteins, a reaction named glycation. Pre-clinical reports show that alpha-synuclein is a target of glycation, and glycation potentiates its pathogenicity which contributes for the neurodegenerative process. Moreover, it triggers, anticipates, or aggravates several PD-like motor and non-motor complications. A given profile of proteins are differently glycated in diseased conditions, altering the brain proteome and leading to brain dysfunction and neurodegeneration. Herein we coin the term Glycatome as the profile of glycated proteins. In this review we report on the mechanisms underlying the association between T2DM and PD, with particular focus on the impact of protein glycation.publishersversionpublishe

Artrite reumatoide: impacto da terapêutica com agentes biológicos nos parâmetros clínicos e laboratoriais

A Artrite Reumatoide (AR) é uma doença autoimune, inflamatória crónica sistémica, de etiologia desconhecida e está associada à incapacidade progressiva, complicações sistémicas, morte prematura e elevados custos socioeconómicos. O tratamento farmacológico da AR pode ser realizado em monoterapia ou em combinação e inclui o uso de anti-inflamatórios não-esteroides, glucocorticoides e fármacos antirreumáticos modificadores da evolução da doença. Nas últimas décadas surgiram os fármacos biológicos e estes vieram revolucionar o tratamento das doenças autoimunes, nomeadamente a AR. Este projeto de investigação é num estudo retrospetivo desde o ano de 2008, até ao presente ano e a amostra inicial todos os doentes com AR, seguidos nas Consultas de Doenças Autoimunes da Unidade Local de Saúde da Guarda. Assim, o presente trabalho teve como objetivo analisar o impacto da terapêutica com agentes biológicos no Disease Activity Score (DAS28) e em diferentes parâmetros laboratoriais em doentes com AR. Procedeu-se a uma análise dos perfis de evolução da doença e das correlações entre o DAS28 e diferentes parâmetros laboratoriais. Os parâmetros laboratoriais foram analisados no Laboratório de Análises Clínicas do Serviço de Patologia Clínica da Unidade Local de Saúde da Guarda de acordo com as normas e critérios definidos para a avaliação de cada parâmetro. Concluiu-se que a terapêutica convencional e a terapêutica biológica apresentam uma eficácia semelhante, nomeadamente no que diz respeito ao DAS28 e também à generalidade dos parâmetros laboratoriais avaliados. No entanto, no que diz respeito à sintomatologia, o tratamento com fármacos biológicos conduz a melhorias significativas, com um aumento global da qualidade de vida dos doentes. O estudo da evolução do DAS28 e de parâmetros laboratoriais ao longo do tempo permitiu observar a variabilidade interindividual existente nos doentes com AR, o que pressupõe a prática de uma “medicina personalizada”. No que diz respeito à correlação entre parâmetros observou-se que existe uma correlação positiva entre o DAS28 e a velocidade de sedimentação e uma correlação negativa entre o DAS28 e a hemoglobina nos grupos analisados. Relativamente aos parâmetros imunológicos e à sua correlação com o DAS28, os resultados sugerem que estes parâmetros não apresentam capacidade de prognóstico da doença e monitorização da terapêutica. Em suma, do ponto de vista dos parâmetros laboratoriais e do DAS28 não se observa um impacto significativo do tratamento com fármacos biológicos.Rheumatoid Arthritis (RA) is a chronic systemic inflammatory autoimmune disease, with unknown etiology and it is associated with progressive disability, systemic complications, early death and high socioeconomic costs. Pharmacological treatment of RA can be performed alone or in combination and includes the use of nonsteroidal anti-inflammatory drugs, glucocorticoids and disease-modifying antirheumatic drugs. In the latest decades, biological drugs have emerged and these drugs have revolutionized the treatment of autoimmune diseases, particular RA. This research project was a retrospective study from the year 2008 until the present year and the initial sample have included all RA patients, followed in Consultas de Doenças Autoimunes da Unidade Local de Saúde da Guarda. Therefore, the present study aimed to analyze the impact of therapy with biological agents in the Disease Activity Score (DAS28) and in different laboratory parameters in patients with RA. Additionally we performed an analysis of the profiles of the disease and correlations between DAS28 and various laboratory parameters. Laboratory parameters were analyzed in the Laboratório de Análises Clínicas do Serviço de Patologia Clínica da Unidade Local de Saúde da Guarda according to the standards and criteria for the evaluation of each parameter. We concluded that conventional therapy and biological therapy have similar efficacy, particularly with regard to the DAS28 and also to most of the laboratory parameters. However, with regard to the physical symptoms of RA, treatment with biological agents leads to significant improvement, with an overall increase in the quality of life of patients. The detailed study of the evolution of the DAS28 and laboratory parameters showed the interindividual variability in patients with RA, which requires the commission of a "personalized medicine". With regard to the correlation between the parameters, we observed that there is a positive correlation between the DAS28 and sedimentation and a negative correlation between the DAS28 and haemoglobin in the control group and study group, respectively. Regarding the immunological parameters and their correlation with DAS28, the results suggest that these parameters do not have the ability to prognosis and monitoring of therapy. In summary, from the viewpoint of laboratory parameters and DAS28 there is not observed a significant impact of the treatment with biological agents. However, there is a growing need for finding a predictive biomarker which allows to efficient monitoring of the therapy

Trick or Treat?

Funding Information: Funding: AC and FOM were supported by grants and contracts from the Portuguese Foundation for Science and Technology, PD/BD/136863/2018 and CEECIND/04266/2017, respectively.Accumulating evidence suggests the existence of a strong link between metabolic syndrome and neurodegeneration. Indeed, epidemiologic studies have described solid associations between metabolic syndrome and neurodegeneration, whereas animal models contributed for the clarification of the mechanistic underlying the complex relationships between these conditions, having the development of an insulin resistance state a pivotal role in this relationship. Herein, we review in a concise manner the association between metabolic syndrome and neurodegeneration. We start by providing concepts regarding the role of insulin and insulin signaling pathways as well as the pathophysiological mechanisms that are in the genesis of metabolic diseases. Then, we focus on the role of insulin in the brain, with special attention to its function in the regulation of brain glucose metabolism, feeding, and cognition. Moreover, we extensively report on the association between neurodegeneration and metabolic diseases, with a particular emphasis on the evidence observed in animal models of dysmetabolism induced by hypercaloric diets. We also debate on strategies to prevent and/or delay neurodegeneration through the normalization of whole-body glucose homeostasis, particularly via the modulation of the carotid bodies, organs known to be key in connecting the periphery with the brain.publishersversionpublishe

Underlying role of glycation in the pathogenesis of Parkinson’s disease

Parkinson’s disease (PD) is the most common movement disorder and the second most common neurodegenerative disorder, affecting 7 to 10 million people worldwide and is an age-related disease. PD is characterized by a progressive motor decline that includes bradykinesia and tremor, and by non-motor features such as mood and cognitive changes and belongs to a wide group of neurodegenerative diseases known as synucleinopathies. The neuropathological hallmarks of PD are the presence of intracellular proteinaceous aggregates primarily composed of alpha-synuclein (aSyn), known as Lewy bodies (LBs) and Lewy neurites (LNs), and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) within the midbrain. The degeneration of dopaminergic neurons in the SNpc leads to the depletion of dopamine in the striatum that underlies the cardinal motor features of parkinsonism observed in PD. Parkinsonism is the clinical syndrome characterized by resting tremors, bradykinesia, muscular rigidity, postural instability, and gait impairment. Although the dopaminergic system in the SNpc is highly affected, several other neurotransmission and neuromodulatory systems are also implicated in PD including the glutamatergic, GABAergic, and cholinergic pathways. The imbalance of these neurotransmission systems impacts both motor and non-motor features of PD. Although several genetic mutations and polymorphisms have been associated with familial forms of PD, about 90% of the cases are sporadic or idiopathic. Currently, PD lacks curative, restorative, preventive, or disease-modifying therapies and this is one of the major challenges in the field. The available therapeutic approaches and tools for PD are restricted to the symptomatic management of motor features and other non-motor signs. aSyn is a small presynaptic protein, abundant in the brain and in several other tissues. aSyn has been reported to participate in synaptic vesicles (SV) dynamics and in different steps of neurotransmitters release. aSyn is a protein that is natively unfolded and highly prone to aggregation. The oligomerization and aggregation of aSyn is believed to contribute for the neurodegenerative process in PD and related synucleinopathies. Although valuable information about the mechanisms governing aSyn pathogenicity and aggregation have been collected from the study of PD-linked mutations, the pathological mechanisms in PD remain largely elusive as most cases are sporadic. Our poor understanding of the molecular mechanisms underlying neurodegeneration in PD has been one of the major obstacles for the development of effective therapies. Although ageing is the major risk factor for neurodegenerative diseases, such as PD, it is imperative to identify and better understand the role and contribution of different risk factors for the pathogenesis of PD, as well as to disclose the mechanisms driving to protein abnormalities and aggregation. In the last 20 years, diabetes mellitus (DM) emerged as a relevant risk factor for neurodegenerative diseases such as PD. Epidemiological studies revealed that type 2 diabetes mellitus (T2DM) can increase up to 40% the risk of developing PD in the general population, and this risk rises to 380% for young diabetic individuals. T2DM is a chronic metabolic disease characterized by glucose metabolism imbalance, hyperglycaemia, insulin resistance, and impaired glucose tolerance. Hyperglycaemia is a hallmark of T2DM and is the major responsible for the deleterious accumulation of the highly reactive reducing sugars, such as methylglyoxal (MGO), that are unavoidably formed as by-products of glycolysis. MGO, one of the most powerful reducing sugars, reacts and covalently bind to biomolecules such as proteins in a non-enzymatic process named glycation, leading to the formation of the irreversible advanced glycation end products (AGEs). Compelling evidence support a key role of glycation in the aetiology, pathogenesis, and progression of PD. Studies reported the presence of AGEs at the periphery of LBs, increased levels of glycation in the brains of patients with PD, and of glycated aSyn in the brains of patients with PD and FTD. In vitro studies showed that glycation accelerates the formation of aSyn oligomers by impairing both the proteasome and lysosome activity, contributing to the loss of proteostasis, and promoting neuronal cell death. In a transgenic mouse model of aSyn pathology, MGO injection in the substantia nigra or striatum induced neurodegeneration, particularly of dopaminergic neurons. Considering the prior findings suggesting a pivotal role of glycation in potentiating aSyn pathology, our main hypothesis is that glycation triggers or exacerbates PD-like phenotypes. Furthermore, we also hypothesize that strategies aimed at suppressing glycation may prove effective in preventing PD onset and progression. To unveil the brain proteome response to aSyn pathology and the role of protein glycation on aSyn pathobiology in the brain, age-matched male transgenic Thy1-aSyn and WT littermates mice received a single dose of MGO through intracerebroventricular (ICV) injection. Three weeks after the procedure, mice were weighed, handled, and the Shirpa protocol performed. Behavioural phenotyping was performed four weeks after surgery to evaluate motor, cognitive, olfactory, and anxiety-related function. Here, we reported that 20 weeks Thy1-aSyn mice exhibited impaired motor performance comparing to WT littermates. In this time-point, we did not observe colonic dysfunction, olfactory alterations, cognitive impairment, neither anxiety-related alterations. Thy1-aSyn mice at 20 weeks of age did not exhibit dopaminergic or non-dopaminergic neuronal degeneration in the SNpc. By using a quantitative SWATH proteomics approach, we disclosed the pathological impact of aSyn overexpression in the brain of the transgenic Thy1-aSyn mouse model by defining the molecular targets and associated dysregulated pathways. We found that aSyn overexpression in the midbrain mostly impacts proteins associated with oxidative phosphorylation, endocytosis and intracellular vesicular trafficking, and synaptic vesicles dynamics. Although this work does not unveil new pathways implicated in aSyn pathology, it clearly identifies the molecular targets that are dysregulated upon aSyn overexpression in the midbrain. By determining the profile of proteins responding to aSyn pathology, we can unveil potential molecular targets that might hold potential as aSyn pathology-modifiers, which can disclose new therapeutic avenues for synucleinopathies. In this work, we also hypothesized that glycation-induced neuronal dysfunction might be a contributing factor in synucleinopathies such as PD. We reported that MGO-glycation in the brain potentiates motor, cognitive, olfactory, and colonic dysfunction in transgenic Thy1-aSyn mice that received a single dose of MGO by ICV injection. In these mice, aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. We also observed increased aSyn phosphorylation at S129 and aSyn insolubility in the midbrain. MGO triggered a general neuronal loss at the vicinity of SNpc within the midbrain. SWATH mass spectrometry analysis disclosed that glycation mainly increase glutamatergic signalling associated proteins in the midbrain. By contrast, in the prefrontal cortex the most affected proteins correspond to the electron transport chain. These findings suggest that MGO differently impacts the proteome depending on the brain region. Furthermore, glycated proteins in the midbrain of MGO-injected Thy1-aSyn mice strongly correlate with PD and dopaminergic pathways. Altogether, our study demonstrated that MGO-induced glycation accelerates PD-like sensorimotor and cognitive alterations, pointing that an increased glutamatergic signalling may underly the exacerbated behaviour phenotype. Our study not only sheds new light into the enhanced vulnerability of the midbrain in PD-related synaptic dysfunction, but also defines a link between PD and T2DM. Moreover, our results support that glycation suppressors and anti-glutamatergic drugs may hold promise as disease-modifying therapies for PD and related synucleinopathies. Considering the pivotal role of glycation on aSyn pathobiology and in the pathogenesis and progression of PD, we hypothesize that strategies aimed at blocking or suppressing glycation could be used as novel preventive or disease-modifying therapies for PD and other synucleinopathies. To test this hypothesis, we used both a genetic strategy and a drug-based screening of compounds that target glycation. Following the observation that a MGO insult in H4 neuroglioma cells decreases the levels of heat shock protein 27 (Hsp27) in a dose-dependent manner, we hypothesized that compensating Hsp27 loss in glycating environments could alleviate the glycation-induced pathogenicity of aSyn. In this cellular system, we co- expressed aSyn and Hsp27 and after challenging these cells to MGO glycation we found that Hsp27 overexpression prevented MGO-induced aSyn cytotoxicity, accumulation, and aggregation. Our work uncovers the importance of Hsp27 in suppressing the pathological conditions induced by glycation, suggesting that Hsp27 may constitute a suitable target for intervention in PD and related synucleinopathies. By using a drug-based screening, H4 neuroglioma cells overexpressing aSyn or SynT under glycating conditions were treated with different compounds. From the 11 compounds tested we found that carnosine, metformin, and penicillamine prevented MGO-induced aSyn pathology. Metformin prevented MGO- associated aSyn cytotoxicity, SynT insolubility and aggregation, and the impairment of aSyn clearance. Carnosine prevented MGO-associated aSyn cytotoxicity, SynT insolubility and inclusions formation. Penicillamine prevented MGO-induced cytotoxicity, the increase in the levels of aSyn and SynT levels, SynT insolubility and aggregation and improved aSyn clearance. Our findings suggest that carnosine, metformin, and penicillamine are the most promising and lead therapeutic compounds requiring further validation in pre-clinical models of synucleinopathies. There is an urgent demand for preventive or disease-modifying therapies for PD and other neurodegenerative diseases. Our work highlights the pivotal role of glycation in aSyn pathobiology and in the exacerbation of PD-like features, narrowing the bridge between PD and DM. Our data suggests decreasing glycation and targeting the glutamatergic system hold preventive or disease-modifying potential therapies for PD and other synucleinopathies. Our research opens novel therapeutic avenues for synucleinopathies that needs to be properly exploited in both pre-clinical and clinical studies

Coexistence between transgenic MON 810 maize and hives: pollen and flour flow by pollinator bees and honey labelling

In Portugal, in 2012 and 2013, the cultivation of GM maize covered 9278 ha and 8202.2 ha, respectively, representing approximately 6-7% of the total maize sowing areas. The large-scale cultivation of GM maize is not uniformly distributed along the country. It has its highest expression in the Centre-South regions. Maize is a cross-pollinating species and, preferentially, its pollen is wind-dispersed although it may also be insect-dispersed. In 2011, the European Court of Justice decided upon the need to demonstrate that pollen is a natural constituent of honey rather than an ingredient. Later, in 2013, the European Parliament defined pollen as a natural constituent of honey. This decision avoided strong financial implications concerning the need of honey labelling whenever Genetically Modified (GM) pollen makes up more than 0.9% of the species pollen fraction, according to the Regulation (EC) 1829/2003. Contrarily, the presence of flour, which is not a natural component of honey but may occur when industrial mills are in the neighborhood of the hives, might be treated according to the legal labelling framework for ingredients. Therefore, there is a need to distinguish between pollen and flour. Currently, the detection/quantification of GM components in honey is done by real-time PCR. However, until now no procedure can distinguish pollen from other kinds of GM material. Our main goal was, therefore, to develop a reliable and accurate method to allow distinguishing pollen from flour in honey. We investigated the ability of quantitative real-time PCR together with plasmid calibrants, triploid maize seed endosperms and haploid maize pollen, to develop a new approach to differentiate between adventitious presence of GM pollen and adventitious presence of GM flour. Plasmid calibrant certified for a 1:1 copy number ratio (transgene copy number in relation to an endogenous gene copy number) allow for the distinction between triploid and haploid tissues. A seven point dilution series from a 2x106 plasmid copies/μL solution was used to establish two calibration curves, being one for the transgene and other for a species-specific gene. DNA was extracted from honey, originally obtained in a Portuguese GM free zone (Região Autónoma da Madeira), spiked either with pollen, flour or embryos from a hemizygous population having the female progenitor as the transgene donor. PCR efficiencies were of 93 and 94% for both reactions. As proof-of-concept, samples of national and imported honey, commercialized either at local producers or in the supermarkets, were analyzed and the quantification results compared with the spiked honey samples. With this information available, correct estimation of the relative transgene copy number allowed for the distinction between pollen and flour. We have adapted real-time PCR to fit into the requirements of GMO labelling regulations. This approach has considerable potential to evaluate escapes of maize flour and for the establishment of recommendations for the milling companies in order to minimize effective entry of flour into the hives. The procedure has been in-house validated for maize

Posttranslational modifications of blood-derived alpha-synuclein as biochemical markers for Parkinson’s disease

Abstract Parkinson’s disease (PD) is a progressive neurodegenerative disorder known for the typical motor features associated. Pathologically, it is characterized by the intracellular accumulation of alpha-synuclein (aSyn) in Lewy bodies and Lewy neurites. Currently, there are no established biochemical markers for diagnosing or for following disease progression, a major limitation for the clinical practice. Posttranslational modifications (PTMs) in aSyn have been identified and implicated on its pathobiology. Since aSyn is abundant in blood erythrocytes, we aimed to evaluate whether PTMs of aSyn in the blood might hold value as a biomarker for PD. We examined 58 patients with PD and 30 healthy age-matched individuals. We found that the levels of Y125 phosphorylated, Y39 nitrated, and glycated aSyn were increased in PD, while those of SUMO were reduced. A combinatory analysis of the levels of these PTMs resulted in an increased sensitivity, with an area under curve (AUC) of 0.843 for PD versus healthy controls, and correlated with disease severity and duration. We conclude that the levels of these selected PTMs hold strong potential as biochemical markers for PD. Ultimately, our findings might facilitate the monitoring of disease progression in clinical trials, opening the possibility for developing more effective therapies against PD

Glycation modulates glutamatergic signaling and exacerbates Parkinson’s disease-like phenotypes

Funding Information: This study was supported by Fundação para a Ciência e Tecnologia (FCT) PTDC/NEU-OSD/5644/2014, by iNOVA4Health UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by FCT/Ministério da Ciência, Tecnologia e Ensino Superior, through national funds; and by Sociedade Portuguesa de Diabetologia. The authors were supported by: A.C. (FCT, PD/BD/136863/2018; ProRegeM – PhD programme, mechanisms of disease and regenerative medicine); B.F.G. (PTDC/NEU-OSD/5644/2014); L.S. (SFRH/BD/143286/2019). T.F.O. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy - EXC 2067/1- 390729940, and by SFB1286 (B8). Funding Information: We would like to thank the vivarium and behavioral facilities at Instituto de Medicina Molecular?Jo?o Lobo Antunes for all the support. We also thank Prof. Rosalina Fonseca, Prof. S?lvia V. Conde, Dr Rita Machado de Oliveira, Dr Nat?lia Madeira, Dr Liliana Lopes, Dr Tatiana Burrinha, and Dr Catarina Perdig?o for fruitful discussions. We thank Dr Manuela Correia for all the laboratory support. We are deeply thankful to Prof. Jos? Ramalho for kindly allowing the use of equipment for protein analysis. This study was supported by Funda??o para a Ci?ncia e Tecnologia (FCT) PTDC/NEU-OSD/5644/2014, by iNOVA4Health UIDB/04462/2020 and UIDP/04462/2020, a program financially supported by FCT/Minist?rio da Ci?ncia, Tecnologia e Ensino Superior, through national funds; and by Sociedade Portuguesa de Diabetologia. The authors were supported by: A.C. (FCT, PD/BD/136863/2018; ProRegeM ? PhD programme, mechanisms of disease and regenerative medicine); B.F.G. (PTDC/NEU-OSD/5644/2014); L.S. (SFRH/BD/143286/2019). T.F.O. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany?s Excellence Strategy - EXC 2067/1- 390729940, and by SFB1286 (B8). Publisher Copyright: © 2022, The Author(s).Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson’s disease (PD). Interestingly, a common molecular alteration among these disorders is the age-associated increase in protein glycation. We hypothesized that glycation-induced neuronal dysfunction is a contributing factor in synucleinopathies. Here, we dissected the impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain. We found that MGO-glycation potentiates motor, cognitive, olfactory, and colonic dysfunction in aSyn transgenic (Thy1-aSyn) mice that received a single dose of MGO via intracerebroventricular injection. aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. SWATH mass spectrometry analysis, used to quantify changes in the brain proteome, revealed that MGO mainly increase glutamatergic-associated proteins in the midbrain (NMDA, AMPA, glutaminase, VGLUT and EAAT1), but not in the prefrontal cortex, where it mainly affects the electron transport chain. The glycated proteins in the midbrain of MGO-injected Thy1-aSyn mice strongly correlate with PD and dopaminergic pathways. Overall, we demonstrated that MGO-induced glycation accelerates PD-like sensorimotor and cognitive alterations and suggest that the increase of glutamatergic signaling may underly these events. Our study sheds new light into the enhanced vulnerability of the midbrain in PD-related synaptic dysfunction and suggests that glycation suppressors and anti-glutamatergic drugs may hold promise as disease-modifying therapies for synucleinopathies.publishersversionpublishe