Altered expression of Sialyl Lewis X in experimental models of Parkinson's disease.

The mechanisms underlying neurodegeneration in Parkinson's disease (PD) are still not fully understood. Glycosylation is an important post-translational modification that affects protein function, cell-cell contacts and inflammation and can be modified in pathologic conditions. Although the involvement of aberrant glycosylation has been proposed for PD, the knowledge of the diversity of glycans and their role in PD is still minimal. Sialyl Lewis X (sLeX) is a sialylated and fucosylated tetrasaccharide with essential roles in cell-to-cell recognition processes. Pathological conditions and pro-inflammatory mediators can up-regulate sLeX expression on cell surfaces, which has important consequences in intracellular signalling and immune function. Here, we investigated the expression of this glycan using in vivo and in vitro models of PD. We show the activation of deleterious glycation-related pathways in mouse striatum upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin-based model of PD. Importantly, our results show that MPTP triggers the presentation of more proteins decorated with sLeX in mouse cortex and striatum in a time-dependent manner, as well as increased mRNA expression of its rate-limiting enzyme fucosyltransferase 7. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. Although the underlying mechanism that drives increased sLeX epitopes, the nature of the protein scaffolds and their functional importance in PD remain unknown, our data suggest for the first time that sLeX in the brain may have a role in neuronal signalling and immunomodulation in pathological conditions. KEY MESSAGES: MPTP triggers the presentation of proteins decorated with sLeX in mouse brain. MPTP triggers the expression of sLeX rate-limiting enzyme FUT 7 in striatum. sLeX is expressed in neurons, including dopaminergic neurons, and microglia. sLeX in the brain may have a role in neuronal signalling and immunomodulation.

NPC1-like phenotype, with intracellular cholesterol accumulation and altered mTORC1 signaling in models of Parkinson's disease.

Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP+), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP+ treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP+. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP+. Together with the MPP+-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP+ insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP+ decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.

Cholesterol redistribution triggered by CYP46A1 gene therapy improves major hallmarks of Niemann-Pick type C disease but is not sufficient to halt neurodegeneration.

Cholesterol 24-hydroxylase (CYP46A1) is an exclusively neuronal cytochrome P450 enzyme responsible for converting cholesterol into 24S-hydroxycholesterol, which serves as the primary pathway for eliminating cholesterol in the brain. We and others have shown that increased activity of CYP46A1 leads to reduced levels of cholesterol and has a positive effect on cognition. Therefore, we hypothesized that CYP46A1 could be a potential therapeutic target in Niemann-Pick type C (NPC) disease, a rare and fatal neurodegenerative disorder, characterized by cholesterol accumulation in endolysosomal compartments. Herein, we show that CYP46A1 ectopic expression, in cellular models of NPC and in Npc1tm(I1061T) mice by adeno-associated virus-mediated gene therapy improved NPC disease phenotype. Amelioration in functional, biochemical, molecular and neuropathological hallmarks of NPC disease were characterized. In vivo, CYP46A1 expression partially prevented weight loss and hepatomegaly, corrected the expression levels of genes involved in cholesterol homeostasis, and promoted a redistribution of brain cholesterol accumulated in late endosomes/lysosomes. Moreover, concomitant with the amelioration of cholesterol metabolism dysregulation, CYP46A1 attenuated microgliosis and lysosomal dysfunction in mouse cerebellum, favoring a pro-resolving phenotype. In vivo CYP46A1 ectopic expression improves important features of NPC disease and may represent a valid therapeutic approach to be used concomitantly with other drugs. However, promoting cholesterol redistribution does not appear to be enough to prevent Purkinje neuronal death in the cerebellum. This indicates that cholesterol buildup in neurons might not be the main cause of neurodegeneration in this human lipidosis.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Revisiting Proteasome Inhibitors: Molecular Underpinnings of Their Development, Mechanisms of Resistance and Strategies to Overcome Anti-Cancer Drug Resistance.

Proteasome inhibitors have shown relevant clinical activity in several hematological malignancies, namely in multiple myeloma and mantle cell lymphoma, improving patient outcomes such as survival and quality of life, when compared with other therapies. However, initial response to the therapy is a challenge as most patients show an innate resistance to proteasome inhibitors, and those that respond to the therapy usually develop late relapses suggesting the development of acquired resistance. The mechanisms of resistance to proteasome inhibition are still controversial and scarce in the literature. In this review, we discuss the development of proteasome inhibitors and the mechanisms of innate and acquired resistance to their activity-a major challenge in preclinical and clinical therapeutics. An improved understanding of these mechanisms is crucial to guiding the design of new and more effective drugs to tackle these devastating diseases. In addition, we provide a comprehensive overview of proteasome inhibitors used in combination with other chemotherapeutic agents, as this is a key strategy to combat resistance.

Autocuidado nas pessoas idosas com diabetes mellitus tipo 2: intervenções promotoras de atividade física e alimentação saudável / Self-care in the elderly with type 2 diabetes mellitus: interventions that promote physical activity and healthy eating

Introdução: Atualmente existe um aumento progressivo da incidência e prevalência da Diabetes Mellitus tipo 2. Condição de saúde relevante para o envelhecimento da população, aproximadamente um quarto das pessoas idosas são portadoras da doença, considerada uma das doenças crónicas prioritárias na intervenção a nível mundial. Os comportamentos de autocuidado e a gestão da doença são um suporte fundamental para a pessoa. Integra assim um papel ativo no tratamento e controlo da sua doença, mudando comportamentos e adotando estilos de vida saudável, fatores determinantes para a manutenção do seu bem-estar e qualidade de vida. Os Cuidados de Saúde Primários assumem um papel fundamental na educação das pessoas com diabetes, mobilizando estratégias que dêem resposta às necessidades identificadas, capacitando-as para o autocuidado e promovendo a gestão da sua doença. Objetivo: Promover a adesão do autocuidado a pessoas idosas com Diabetes Mellitus tipo 2, relativamente á atividade física e á alimentação saudável adaptada á sua condição de doença, na USF Dafundo de Outubro de 2019 a Fevereiro de 2020. Metodologia: Utilizamos a metodologia do Processo de Planeamento em Saúde e a Teoria de Enfermagem do Deficit do Autocuidado de Dorothea Orem, numa amostra de pessoas com 65 e mais anos. Aplicamos a todos os participantes através da entrevista, a avaliação dos dados sociodemográficos, Escala de Barthel, Escala de Lawton & Brody e a Escala de Atividades de Autocuidado com a Diabetes. Resultados: Identificamos défices de autocuidado nas pessoas com diabetes pela não adesão á atividade física e a uma alimentação saudável. O processo de capacitação foi promovido através da educação para a saúde individual e em grupo por possibilitar a aquisição individualizado por um lado e a partilha conhecimentos/competências e experiencias vivenciadas, capacitando a pessoa a idosa para a promoção do autocuidado.

Introduction: Currently there is a progressive increase in the incidence and prevalence of type 2 Diabetes Mellitus. A relevant health condition for the aging of the population, approximately one quarter of the elderly are carriers of the disease, considered one of the priority chronic diseases in global intervention. Self-care behaviors and disease management are a fundamental support for the person. It thus integrates an active role in the treatment and control of your disease, changing behaviors and adopting healthy lifestyles, determining factors for the maintenance of their well-being and quality of life. Primary Health Care plays a key role in educating people with diabetes, mobilizing strategies that respond to the identified needs, empowering them for self-care and promoting the management of their disease. Objective: To promote adherence to self-care for elderly people with type 2 diabetes mellitus, physical activity and healthy eating adapted to their disease condition, at the USF Dafundo from October 2019 to February 2020. Methodology: We used the methodology of the Health Planning Process and Dorothea Orem's Self-Care Deficit Nursing Theory, in a sample of people aged 65 and over. We apply to all participants through the interview, the evaluation of sociodemographic data, Barthel Scale, Lawton & Brody Scale and The Diabetes SelfCare Activity Scale. Results: We identified self-care deficits in people with diabetes due to nonadherence to physical activity and healthy eating. The training process was promoted through education for individual and group health, as it enables individualized acquisition on the one hand and the sharing of knowledge / skills and experiences, enabling the elderly person to promote self-care.

Neurotoxic effects of MPTP on mouse cerebral cortex: Modulation of neuroinflammation as a neuroprotective strategy.

Parkinson's disease (PD) is a progressive neurological disorder, mainly characterized by the progressive loss of dopaminergic neurons in the Substantia nigra pars compacta (SNpc) and by the presence of intracellular inclusions, known as Lewy bodies. Despite SNpc being considered the primary affected region in PD, the neuropathological features are confined solely to the nigro-striatal axis. With disease progression other brain regions are also affected, namely the cerebral cortex, although the spreading of the neurologic damage to this region is still not completely unraveled. Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid that has been shown to have antioxidant properties and to exhibit a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice model of PD. Moreover, TUDCA anti-inflammatory properties have been reported in glial cells, making it a prominent therapeutic agent in PD. Here, we used C57BL/6 mice injected with MPTP in a sub-acute paradigm aiming to investigate if the neurotoxic effects of MPTP could be extended to the cerebral cortex. In parallel, we evaluated the anti-oxidant, neuroprotective and anti-inflammatory effects of TUDCA. The anti-inflammatory mechanisms elicited by TUDCA were further dissected in microglia cells. Our results show that MPTP leads to a decrease of ATP and activated AMP-activated protein kinase levels in mice cortex, and to a transient increase in the expression of antioxidant downstream targets of nuclear factor erythroid 2 related factor 2 (Nrf-2), and parkin. Notably, MPTP increases pro-inflammatory markers, while down-regulating the expression of the anti-inflammatory protein Annexin-A1 (ANXA1). Importantly, we show that TUDCA treatment prevents the deleterious effects of MPTP, sustains increased levels of antioxidant enzymes and parkin, and most of all negatively modulates neuroinflammation and up-regulates ANXA1 expression. Additionally, results from cellular models using microglia corroborate TUDCA modulation of ANXA1 synthesis, linking inhibition of neuroinflammation and neuroprotection by TUDCA.

Tauroursodeoxycholic Acid Improves Motor Symptoms in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by severe motor symptoms, and currently there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Tauroursodeoxycholic acid (TUDCA) is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD; however, its effect in PD motor symptoms has never been addressed. In the present work, an extensive behavior analysis was performed to better characterize the MPTP model of PD and to evaluate the effects of TUDCA in the prevention/improvement of mice phenotype. MPTP induced significant alterations in general motor performance paradigms, including increased latency in the motor swimming, adhesive removal and pole tests, as well as altered gait, foot dragging, and tremors. TUDCA administration, either before or after MPTP, significantly reduced the swimming latency, improved gait quality, and decreased foot dragging. Importantly, TUDCA was also effective in the prevention of typical parkinsonian symptoms such as spontaneous activity, ability to initiate movement and tremors. Accordingly, TUDCA prevented MPTP-induced decrease of dopaminergic fibers and ATP levels, mitochondrial dysfunction and neuroinflammation. Overall, MPTP-injected mice presented motor symptoms that are aggravated throughout time, resembling human parkinsonism, whereas PD motor symptoms were absent or mild in TUDCA-treated animals, and no aggravation was observed in any parameter. The thorough demonstration of improvement of PD symptoms together with the demonstration of the pathways triggered by TUDCA supports a subsequent clinical trial in humans and future validation of the application of this bile acid in PD.

Novel insights into the antioxidant role of tauroursodeoxycholic acid in experimental models of Parkinson's disease.

Impaired mitochondrial function and generation of reactive oxygen species are deeply implicated in Parkinson's disease progression. Indeed, mutations in genes that affect mitochondrial function account for most of the familial cases of the disease, and post mortem studies in sporadic PD patients brains revealed increased signs of oxidative stress. Moreover, exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I inhibitor, leads to clinical symptoms similar to sporadic PD. The bile acid tauroursodeoxycholic acid (TUDCA) is an anti-apoptotic molecule shown to protect against MPTP-induced neurodegeneration in mice, but the mechanisms involved are still incompletely identified. Herein we used MPTP-treated mice, as well as primary cultures of mice cortical neurons and SH-SY5Y cells treated with MPP+ to investigate the modulation of mitochondrial dysfunction by TUDCA in PD models. We show that TUDCA exerts its neuroprotective role in a parkin-dependent manner. Overall, our results point to the pharmacological up-regulation of mitochondrial turnover by TUDCA as a novel neuroprotective mechanism of this molecule, and contribute to the validation of TUDCA clinical application in PD.