Impact of disease duration on proteomic bioprofile and prognosis in rheumatoid arthritis patients.

OBJECTIVES: Cardiovascular disease worsens the prognosis of rheumatoid arthritis (RA) and vice-versa. Inflammation may be a common pathway for both conditions. It is expected that a longer RA duration leads to a greater inflammatory cumulative exposure burden; however, studies on the association between RA disease duration and outcomes are scarce. Our aim is to compare the characteristics, biomarker expression and outcomes according to the duration of RA. METHODS: Prospective cohort study including 399 RA patients, with detailed clinical, echocardiographic, and proteomic phenotyping that were compared across tertiles of RA disease duration. Cox proportional models were used to study the association of disease duration with cardiovascular outcomes. RESULTS: RA duration tertiles were: tertile 1 with median of 3.2; tertile 2 with median of 8.8; and tertile 3 with median of 21.8 years. Compared to tertile 1, patients in tertile 3 were older, had more erosive disease, more frequent echocardiographic alterations, lower haemoglobin and walked a shorter distance on the 6MWT. Natriuretic peptides, cathepsin L1, galectin 9, matrix metalloproteinase-12, adrenomedullin and tumour necrosis factor receptor 11A were higher in patients with longer disease duration. Compared to patients in tertile 1, those in tertile 3 had higher risk of a subsequent cardiovascular hospitalisation or cardiovascular death (HR 2.71, 95%CI 1.06-6.92, p=0.04). CONCLUSIONS: RA patients with longer disease duration had more organ damage and worse outcomes than those with shorter disease duration. Biomarker expression suggested that patients with longer RA duration had activation of pathways related to inflammation, extracellular matrix organisation, fibrosis and congestion.

Nuclear localization and phosphorylation modulate pathological effects of alpha-synuclein.

Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

A familial ATP13A2 mutation enhances alpha-synuclein aggregation and promotes cell death.

Aberrant protein-protein interactions are a common pathological hallmark among neurodegenerative diseases, including Parkinson's disease (PD). Thus far, mutations in more than 20 genes have been associated with PD. These genes encode for proteins involved in distinct intracellular pathways, complicating our understanding of the precise molecular mechanisms underlying the disease. Recent reports suggested that the endolysosomal protein ATP13A2 can determine the fate of alpha-synuclein (α-Syn), although no consensus has yet been reached on the mechanisms underlying this effect. Here, we describe, for the first time, the deleterious effect arising from the interaction between the ATP13A2 familial mutant Dup22 with α-Syn. We show that this ATP13A2 mutant can enhance α-Syn oligomerization and aggregation in cell culture. Additionally, we report the accumulation of both proteins in abnormal endoplasmic reticulum membranous structures and the activation of the protein kinase RNA-like endoplasmic reticulum kinase pathway. Ultimately, our data bring new insight into the molecular mechanisms underlying the interplay of these two proteins, opening novel perspectives for therapeutic intervention.

Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors.

Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.

Structure, function and toxicity of alpha-synuclein: the Bermuda triangle in synucleinopathies.

Parkinson's disease belongs to a group of currently incurable neurodegenerative disorders characterized by the misfolding and accumulation of alpha-synuclein aggregates that are commonly known as synucleinopathies. Clinically, synucleinopathies are heterogeneous, reflecting the somewhat selective neuronal vulnerability characteristic of each disease. The precise molecular underpinnings of synucleinopathies remain unclear, but the process of aggregation of alpha-synuclein appears as a central event. However, there is still no consensus with respect to the toxic forms of alpha-synuclein, hampering our ability to use the protein as a target for therapeutic intervention. To decipher the molecular bases of synucleinopathies, it is essential to understand the complex triangle formed between the structure, function and toxicity of alpha-synuclein. Recently, important steps have been undertaken to elucidate the role of the protein in both physiological and pathological conditions. Here, we provide an overview of recent findings in the field of alpha-synuclein research, and put forward a new perspective over paradigms that persist in the field. Establishing whether alpha-synuclein has a causative role in all synucleinopathies will enable the identification of targets for the development of novel therapeutic strategies for this devastating group of disorders. Alpha-synuclein is the speculated cornerstone of several neurodegenerative disorders known as Synucleinopathies. Nevertheless, the mechanisms underlying the pathogenic effects of this protein remain unknown. Here, we review the recent findings in the three corners of alpha-synuclein biology - structure, function and toxicity - and discuss the enigmatic roads that have accompanied alpha-synuclein from the beginning. This article is part of a special issue on Parkinson disease.

α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner.

Alpha-synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.

A Rare Cause of Abdominal Pain: IgG4-Related Sclerosing Mesenteritis.

A 67-year-old man with previous cardiovascular disease was referred to our consultation due to a 5-month history of recurrent epigastric pain. Esophagogastroduodenoscopy and full blood workup presented no alterations. CT scan showed an irregularly shaped mass at the root of the mesentery, measuring 40x25x47mm, with spiculated contours and retractile behaviour (a). Simultaneous densification of the adjacent fat and infracentimetric ganglionic formations scattered throughout the mesentery were shown. Surgical biopsy revealed extensive storiform fibrosclerosis, with the presence of interstitial lymphoplasmocytic infiltrate and obliterative phlebitis (b); the plasma cells had mostly IgG expression, with IgG4:IgG ratio >40% (c), accounting for more than 30- 40 IgG4 plasma cells per field. The serum IgG4 level was 137mg/dL. A diagnosis of IgG4-related sclerosing mesenteritis was made, without other organ involvement. Prednisolone (0.6mg/kg/d) improved partially the abdominal pain, so steroid sparing strategy with off-label rituximab was associated. Due to its low prevalence, the understanding of this entity is scarce, and its diagnosis is challenging. Unlike other manifestations of IgG4-related disease, the intra-abdominal disease is identified in later stages, due to unspecific symptoms. This case aims to raise awareness about this condition as a differential diagnosis of abdominal pain.

Esophageal Bullous Pemphigoid.

Bullous pemphigoid is a rare autoimmune dermatologic disease that usually occurs in the elderly. Mucous membrane lesions occur in about 10-35% of patients and are almost always limited to the oral mucous membrane. Esophageal involvement is very rare (4% of cases) and usually presents with chest pain, dysphagia, and odynophagia, though patients are frequently asymptomatic. We report the case of newly diagnosed bullous pemphigoid in a 76-year-old man with a past medical history of dementia. He presented with cutaneous manifestations but also severe gastrointestinal bleeding due to extensive esophageal involvement. Although bullous pemphigoid is mainly a skin disease, mucous membrane lesions should not be overlooked as they are associated with an even poorer outcome. A high index of suspicion for esophageal involvement is needed as its presentation can be fatal, as with our patient. LEARNING POINTS: Bullous pemphigoid is a rare autoimmune disease that should be suspected in elderly patients with itchy cutaneous lesions.Mucous membrane lesions should always be evaluated, as they are associated with a poor prognosis, even if asymptomatic.Early diagnosis should be the main focus, as steroids, the mainstay of treatment, may not be effective in severe cases.

Clinical associations and prognostic implications of 6-minute walk test in rheumatoid arthritis.

The clinical associations and prognostic implications of the 6-minute walk test (6MWT) distance in patients with rheumatoid arthritis (RA) is yet to be explored. To identify the clinical features and prognostic implications associated with the 6MWT in patients with RA. Cohort study including 387 RA patients who underwent 6MWT. Regression models (linear and logistic) were built to identify independent predictors of shorter 6MWT distance. Cox proportional models were used to study the association of 6MWT distance with cardiovascular outcomes. Patients were subdivided according to 6MWT tertiles: 126 patients walked > 405 m, 129 walked 345-405 m, and 132 walked < 345 m. Older age (> 55 years), elevated waist circumference, NT-pro BNP > 125 pg/mL, anemia, C-reactive protein ≥ 3 mg/dL, and troponin T ≥ 14 pg/mL were independent predictors of walking shorter distances. Patients walking less than 345 m had higher risk of a subsequent cardiovascular hospitalization or cardiovascular death compared with patients walking 345 m or more (adjusted HR: 2.98, 95%CI: 1.37-6.51, p = 0.006). Older age, abdominal obesity, anemia, cardiac dysfunction, and inflammation were associated with walking shorter distances in patients with RA. Walking less than 345 m in the 6MWT was associated with a poor cardiovascular prognosis. The 6MWT is simple, reproducible, and inexpensive, easily performed in routine practice, and provides important information regarding the patients´ status and outcomes, enabling the monitorization of the therapeutic optimization of the various domains of the RA.

[Intrahospital Protocol for the Management of COVID-19 Disease in Adults]. / Protocolo Intra-Hospitalar para Abordagem da Doença COVID-19 no Adulto.

The COVID-19 pandemic is currently responsible for over 526 million infections and over 6.3 million deaths. As a new disease, the number of papers on the subject is extensive, motivating considerable heterogeneity in its approach. Despite some medicines having sound evidence of benefit, new interventions and strategies continue to be proposed, and some still lack scientific evidence, which hinders a uniform and consensual approach. This article aims to standardize healthcare to adult patients with moderate-to-critical COVID-19, from the emergency department to hospitalization, either in a general ward or in level 2 or level 3 intensive care units, based on the best and most updated scientific evidence available. This protocol presents recommendations for the stratification of adult patients with COVID-19 disease, adequate workup at admission and during hospitalization, inpatient treatment criteria, general treatment measures, pharmacological treatment, management of complications such as organizing pneumonia and bacterial superinfection, thromboprophylaxis, special considerations on pregnancy and breastfeeding and possible future therapies.

A pandemia de COVID-19 é, atualmente, responsável por mais de 526 milhões de infeções e mais de 6,3 milhões de mortes. Como nova doença, é extenso o número de publicações sobre o tema, motivando uma considerável heterogeneidade na sua abordagem. Apesar de existirem terapêuticas com benefício comprovado, continuam a ser propostas novas intervenções e estratégias, algumas das quais carecendo ainda de suporte científico, dificultando assim uma abordagem uniforme e consensual. Este documento tem como objetivo uniformizar, baseando-se na melhor e mais atualizada evidência científica disponível, a prestação de cuidados aos doentes adultos com COVID-19 moderada a crítica, desde o serviço de urgência até à hospitalização, quer em enfermarias gerais, quer em enfermarias de cuidados intensivos de nível 2 e 3. Este protocolo apresenta recomendações para a estratificação da doença COVID-19, critérios de hospitalização, meios complementares de diagnóstico adequados à admissão e durante a hospitalização, medidas terapêuticas gerais e terapêutica farmacológica dirigida, gestão de complicações como pneumonia organizativa e sobreinfeção bacteriana, tromboprofilaxia, considerações especiais na gravidez e amamentação, e possíveis opções terapêuticas futuras.

Sex differences in circulating proteins of patients with rheumatoid arthritis: A cohort study.

OBJECTIVES: Differences in proteomic profiles between men and women may provide insights into the biological pathways that contribute to known sex differences in rheumatoid arthritis (RA). Studies focusing on sex differences in circulating proteins in RA patients are scarce. Our objective was to investigate the sex differences in circulating proteins of RA patients. METHODS: Cohort study enrolling 399 RA patients. Ninety-four circulating protein-biomarkers (92CVDIIOlink®  + troponin-T + C-reactive protein) were measured. Clinical, demographic, and echocardiographic characteristics were compared between men and women. Sex differences in biomarker expression were assessed using regression modeling. RESULTS: In all, 306 (76.7%) patients were women. Compared with men, women had less visceral fat, smoked less, had diabetes and chronic obstructive pulmonary disease less frequently, and expressed more fatigue, anxiety, and depression. The association with cardiovascular outcomes did not differ between sexes. After adjusting for potential confounders, women expressed higher levels of circulating proteins related to adipokine signaling and vascular function (eg, leptin and vascular endothelial growth factor), whereas men expressed higher levels of circulating proteins related to extracellular matrix organization and inflammation (eg, matrix metalloproteinase-2 and C-reactive protein). These results were not found in patients without RA. CONCLUSION: Sex differences in circulating proteins reflect distinct pathways implicated in the pathogenesis of RA, including inflammation, adiposity, angiogenesis, and extracellular matrix organization. These findings may help further investigations into factors underlying sex-based differences and allow future studies focused on sex-specific personalized treatment approaches in RA. CLINICALTRIALS: gov ID: NCT03960515.

Refractory Orbital Myositis in Systemic Lupus Erythematosus A Role for Rituximab.

Orbital myositis in systemic lupus erythematosus (SLE) is a rare entity with risk of serious complications. Timely treatment with effective immunosuppressors is desirable. We report a case of a 32-year-old female patient with SLE who presented with an acute ocular pain and extraorbital muscle thickening, consistent with orbital myositis. Association with SLE was made after exclusion of other aetiologies. Due to refractoriness to steroids, off-label rituximab was initiated with clinical and imaging parameter improvement. LEARNING POINTS: Orbital myositis is a rare entity that can be present in some autoimmune diseases or mimic other common ocular conditions, such as orbital cellulitis or thyroid orbitopathy.In SLE, it may be the form of presentation of the disease or the only manifestation of active disease, even without elevation of inflammatory biomarkers.Generally, patients with orbital myositis have an excellent response to high-dose corticosteroid therapy, but the risk of recurrence or refractoriness is not negligible.

Circulating Biomarkers and Cardiac Structure and Function in Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) increases the risk for abnormalities of the cardiac structure and function, which may lead to heart failure (HF). Studying the association between circulating biomarkers and echocardiographic parameters is important to screen patients with RA with a higher risk of cardiac dysfunction. Aim: To study the association between circulating biomarkers and echocardiographic parameters in patients with RA. Methods: Echocardiography was performed in 355 patients with RA from RA Porto cohort and the associations between echocardiographic characteristics and 94 circulating biomarkers were assessed. These associations were also assessed in the Metabolic Road to Diastolic Heart Failure (MEDIA-DHF) [392 patients with HF with preserved ejection fraction (HFpEF)] and the Suivi Temporaire Annuel Non-Invasif de la Santé des Lorrains Assurés Sociaux (STANISLAS) (1,672 healthy population) cohorts. Results: In the RA Porto cohort, mean age was 58 ± 13 years, 23% were males and mean RA duration was 12 ± 10 years. After adjustment and multiple testing correction, left ventricular mass index (LVMi), left atrial volume index (LAVi), and E/e' were independently associated with biomarkers reflecting inflammation [i.e., bone morphogenetic protein 9 (BMP9), pentraxin-related protein 3 (PTX3), tumor necrosis factor receptor superfamily member 11a (TNFRSF11A)], extracellular matrix remodeling [i.e., placental growth factor (PGF)], congestion [i.e., N-terminal pro-brain natriuretic peptide (NT-proBNP), adrenomedullin (ADM)], and myocardial injury (e.g., troponin). Greater LVMi [hazard ratio (HR) (95% CI) per 1 g/m2 = 1.03 (1.02-1.04), p < 0.001], LAVi [HR (95% CI) per 1 ml/m2 = 1.03 (1.01-1.06), p < 0.001], and E/e' [HR (95% CI) per 1 = 1.08 (1.04-1.13), p < 0.001] were associated with higher rates of cardiovascular events. These associations were externally replicated in patients with HFpEF and asymptomatic individuals. Conclusion: Circulating biomarkers reflecting inflammation, extracellular matrix remodeling, congestion, and myocardial injury were associated with underlying alterations of cardiac structure and function. Biomarkers might be used for the screening of cardiac alterations in patients with RA.

Prevalence, risk factors and proteomic bioprofiles associated with heart failure in rheumatoid arthritis: The RA-HF study.

BACKGROUND: Rheumatoid arthritis (RA) patients have high risk of heart failure (HF). AIMS: Identifying the risk factors and mechanistic pathways associated with HF in patients with RA. METHODS: Cohort study enrolling 355 RA patients. HF was defined according to the ESC criteria. 93 circulating protein-biomarkers (91CVDIIOlink®+troponin-T+c-reactive protein) were measured. Regression modeling (multivariate and multivariable) were built and network analyses were performed - based on the identified relevant protein biomarkers. RESULTS: 115 (32.4%) patients fulfilled the ESC criteria for HF, but only 24 (6.8%) had a prior HF diagnosis. Patients with HF were older (67 vs. 55yr), had a longer RA duration (10 vs. 14yr), had more frequently diabetes, hypertension, obesity, dyslipidemia, atrial fibrillation, and ischemic arterial disease. Several protein-biomarkers remained independently associated with HF, the top (FDR1%) were adrenomedullin, placenta-growth-factor, TNF-receptor-11A, and angiotensin-converting-enzyme-2. The networks underlying the expression of these biomarkers pointed towards congestion, apoptosis, inflammation, immune system signaling and RAAS activation as central determinants of HF in RA. Similar HF-associated biomarker-pathways were externally found in patients without RA. Having RA plus HF increased the risk of cardiovascular events compared to RA patients without RF; adjusted-HR (95%CI)=2.37 (1.07-5.30), p=0.034 CONCLUSION: Age, cardiovascular risk factors, and RA duration increase the HF odds in patients with RA. Few RA patients had a correct prior HF diagnosis, but the presence of HF increased the patients` risk. RA patients with HF largely share the mechanistic pathways of HF patients without RA. Randomized HF trials should include patients with RA. CLINICALTRIALS. GOV ID: NCT03960515.

Subclinical ventricular dysfunction in rheumatoid arthritis.

Patients with rheumatoid arthritis (RA) are at higher risk for having underdiagnosed heart failure, however there are no recommendations regarding echocardiographic screening. We aimed to determine the prevalence of subclinical ventricular dysfunction in RA applying current echocardiographic guidelines, its association with patients' characteristics, biomarkers and prognostic parameters and compare the 2016 guidelines to the recommendations from 2009. Prospective study of RA patients without known heart disease, categorized as preserved ventricular function (PVF), systolic dysfunction (SD), isolated diastolic dysfunction (DD) or indeterminate diastolic function (IDF) as per the 2016 echocardiography guidelines-or any ventricular dysfunction (AVD) comprehending the last 3. The median age was 58 years and 78% were females. The majority had PVF (73%), followed by DD (13%), IDF (11%) and SD (4%). Concordance with the 2009 echocardiographic guidelines was low. Compared with PVF, AVD patients were older (65 vs 55 years, p < 0.001), had a higher prevalence of hypertension and dyslipidaemia (56% vs 38%, p = 0.003 and 60% vs 41%, p = 0.002, respectively). In multivariable analysis, age (particularly > 57 years) was the only independent predictor of AVD or DD. AVD was significantly associated with higher NT-proBNP and lower distance in 6-min walk test. There were no significant independent associations between characteristics of RA disease and ventricular function. A total of 17% of RA patients without known cardiovascular disease presented subclinical systolic or diastolic dysfunction, which was associated with older age. The echocardiographic screening may have clinical value in identifying subclinical ventricular dysfunction, especially in older RA patients.

Membrane binding, internalization, and sorting of alpha-synuclein in the cell.

Alpha-synuclein (aSyn) plays a crucial role in Parkinson's disease (PD) and other synucleinopathies, since it misfolds and accumulates in typical proteinaceous inclusions. While the function of aSyn is thought to be related to vesicle binding and trafficking, the precise molecular mechanisms linking aSyn with synucleinopathies are still obscure. aSyn can spread in a prion-like manner between interconnected neurons, contributing to the propagation of the pathology and to the progressive nature of synucleinopathies. Here, we investigated the interaction of aSyn with membranes and trafficking machinery pathways using cellular models of PD that are amenable to detailed molecular analyses. We found that different species of aSyn can enter cells and form high molecular weight species, and that membrane binding properties are important for the internalization of aSyn. Once internalized, aSyn accumulates in intracellular inclusions. Interestingly, we found that internalization is blocked in the presence of dynamin inhibitors (blocked membrane scission), suggesting the involvement of the endocytic pathway in the internalization of aSyn. By screening a pool of small Rab-GTPase proteins (Rabs) which regulate membrane trafficking, we found that internalized aSyn partially colocalized with Rab5A and Rab7. Initially, aSyn accumulated in Rab4A-labelled vesicles and, at later stages, it reached the autophagy-lysosomal pathway (ALP) where it gets degraded. In total, our study emphasizes the importance of membrane binding, not only as part of the normal function but also as an important step in the internalization and subsequent accumulation of aSyn. Importantly, we identified a fundamental role for Rab proteins in the modulation of aSyn processing, clearance and spreading, suggesting that targeting Rab proteins may hold important therapeutic value in PD and other synucleinopathies.

LRRK2 Promotes Tau Accumulation, Aggregation and Release.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are known as the most frequent cause of familial Parkinson's disease (PD), but are also present in sporadic cases. The G2019S-LRRK2 mutation is located in the kinase domain of the protein, and has consistently been reported to promote a gain of kinase function. Several proteins have been reported as LRRK2 substrates and/or interactors, suggesting possible pathways involved in neurodegeneration in PD. Hyperphosphorylated Tau protein accumulates in neurofibrillary tangles, a typical pathological hallmark in Alzheimer's disease and frontotemporal dementia. In addition, it is also frequently found in the brains of PD patients. Although LRRK2 is a kinase, it appears that a putative interaction with Tau is phosphorylation-independent. However, the underlying mechanisms and the cellular consequences of this interaction are still unclear. In this study, we demonstrate an interaction between LRRK2 and Tau and that LRRK2 promotes the accumulation of non-monomeric and high-molecular weight (HMW) Tau species independent of its kinase activity. Interestingly, we found that LRRK2 increases Tau secretion, possibly as a consequence of an impairment of Tau proteasomal degradation. Our data highlight a mechanism through which LRRK2 regulates intracellular Tau levels, contributing to the progression of the pathology caused by the LRRK2-mediated proteasome impairment. In total, our findings suggest that the interplay between LRRK2 and proteasome activity might constitute a valid target for therapeutic intervention in PD.

Fasudil attenuates aggregation of α-synuclein in models of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, yet disease-modifying treatments do not currently exist. Rho-associated protein kinase (ROCK) was recently described as a novel neuroprotective target in PD. Since alpha-synuclein (α-Syn) aggregation is a major hallmark in the pathogenesis of PD, we aimed to evaluate the anti-aggregative potential of pharmacological ROCK inhibition using the isoquinoline derivative Fasudil, a small molecule inhibitor already approved for clinical use in humans. Fasudil treatment significantly reduced α-Syn aggregation in vitro in a H4 cell culture model as well as in a cell-free assay. Nuclear magnetic resonance spectroscopy analysis revealed a direct binding of Fasudil to tyrosine residues Y133 and Y136 in the C-terminal region of α-Syn. Importantly, this binding was shown to be biologically relevant using site-directed mutagenesis of these residues in the cell culture model. Furthermore, we evaluated the impact of long-term Fasudil treatment on α-Syn pathology in vivo in a transgenic mouse model overexpressing human α-Syn bearing the A53T mutation (α-Syn(A53T) mice). Fasudil treatment improved motor and cognitive functions in α-Syn(A53T) mice as determined by Catwalk(TM) gait analysis and novel object recognition (NOR), without apparent side effects. Finally, immunohistochemical analysis revealed a significant reduction of α-Syn pathology in the midbrain of α-Syn(A53T) mice after Fasudil treatment. Our results demonstrate that Fasudil, next to its effects mediated by ROCK-inhibition, directly interacts with α-Syn and attenuates α-Syn pathology. This underscores the translational potential of Fasudil as a disease-modifying drug for the treatment of PD and other synucleinopathies.