The subcellular arrangement of alpha-synuclein proteoforms in the Parkinson's disease brain as revealed by multicolor STED microscopy.

Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)-including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn-accumulate in Lewy bodies (LBs) in different regions of the Parkinson's disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.

The Localization of Alpha-synuclein in the Endocytic Pathway.

Alpha-synuclein (αS) is an intrinsically disordered protein (IDP) that is abundantly present in the brain and is associated with Parkinson's disease (PD). In spite of its abundance and its contribution to PD pathogenesis, the exact cellular function of αS remains largely unknown. The ability of αS to remodel phospholipid model membranes combined with biochemical and cellular studies suggests that αS is involved in endocytosis. To unravel with which route(s) and stage(s) of the endocytic pathway αS is associated, we quantified the colocalization between αS and endocytic marker proteins in differentiated SH-SY5Y neuronal cells, using an object based colocalization analysis. Comparison with randomized data allowed us to discriminate between structural and coincidental colocalizations. A large fraction of the αS positive vesicles colocalizes with caveolin positive vesicles, a smaller fraction colocalizes with EEA1 and Rab7. We find no structural colocalization between αS and clathrin and Rab11 positive vesicles. We conclude that in a physiological context, αS is structurally associated with caveolin dependent membrane vesiculation and is found further along the endocytic pathway, in decreasing amounts, on early and late endosomes. Our results not only shed new light on the function of αS, they also provide a possible link between αS function and vesicle trafficking malfunction in PD.

α-Synuclein evokes NLRP3 inflammasome-mediated IL-1ß secretion from primary human microglia.

Synucleinopathies such as Parkinson's disease (PD) are hallmarked by α-synuclein (α-syn) pathology and neuroinflammation. This neuroinflammation involves activated microglia with increased secretion of interleukin-1ß (IL-1ß). The main driver of IL-1ß secretion from microglia is the NLRP3 inflammasome. A critical link between microglial NLRP3 inflammasome activation and the progression of both α-syn pathology and dopaminergic neurodegeneration has been identified in various PD models in vivo. α-Syn is known to activate the microglial NLRP3 inflammasome in murine models, but its relationship to this inflammasome in human microglia has not been established. In this study, IL-1ß secretion from primary mouse microglia induced by α-syn fibrils was dependent on NLRP3 inflammasome assembly and caspase-1 activity, as previously reported. We show that exposure of primary human microglia to α-syn fibrils also resulted in significant IL-1ß secretion that was dependent on inflammasome assembly and involved the recruitment of caspase-1 protein to inflammasome scaffolds as visualized with superresolution microscopy. While canonical IL-1ß secretion was clearly dependent on caspase-1 enzymatic activity, this activity was less clearly involved for α-syn-induced IL-1ß secretion from human microglia. This work presents similarities between primary human and mouse microglia in the mechanisms of activation of the NLRP3 inflammasome by α-syn, but also highlights evidence to suggest that there may be a difference in the requirement for caspase-1 activity in IL-1ß output. The data represent a novel characterization of PD-related NLRP3 inflammasome activation in primary human microglia and further implicate this mechanism in the pathology underlying PD.

Neoadjuvant cisplatin and paclitaxel modulate tumor-infiltrating T cells in patients with cervical cancer.

Resistance to chemotherapy is widely recognized as one of the major factors limiting therapeutic efficacy and influences clinical outcomes in patients with cancer. Many studies on various tumor types have focused on combining standard-of-care chemotherapy with immunotherapy. However, for cervical cancer, the role of neoadjuvant chemotherapy (NACT) on the local immune microenvironment is largely unexplored. We performed a pilot study on 13 primary cervical tumor samples, before and after NACT, to phenotype and enumerate tumor-infiltrating T-cell subpopulations using multiplex immunohistochemistry (CD3, CD8, FoxP3, Ki67, and Tbet) and automated co-expression analysis software. A significant decrease in proliferating (Ki67+) CD3+CD8- T cells and FoxP3+(CD3+CD8-) regulatory T cells was observed in the tumor stroma after cisplatin and paclitaxel treatment, with increased rates of cytotoxic CD8+ T cells, including activated and CD8+Tbet+ T cells. No effect was observed on the number of tumor-infiltrating T cells in the cervical tumor microenvironment after treatment with cisplatin only. Therefore, we conclude that patients treated with cisplatin and paclitaxel had more tumor-infiltrating T-cell modulation than patients treated with cisplatin monotherapy. These findings enhance our understanding of the immune-modulating effect of chemotherapy and warrant future combination of the standard-of-care therapy with immunotherapy to improve clinical outcome in patients with cervical cancer.

Indoleamine 2,3-Dioxygenase Expression Pattern in the Tumor Microenvironment Predicts Clinical Outcome in Early Stage Cervical Cancer.

The indoleamine 2,3-dioxygenase (IDO) enzyme can act as an immunoregulator by inhibiting T cell function via the degradation of the essential amino acid tryptophan (trp) into kynurenine (kyn) and its derivates. The kyn/trp ratio in serum is a prognostic factor for cervical cancer patients; however, information about the relationship between serum levels and IDO expression in the tumor is lacking. IDO expression was studied in 71 primary and 14 paired metastatic cervical cancer samples by various immunohistochemical (IHC) techniques, including 7-color fluorescent multiparameter IHC, and the link between the concentration of IDO metabolites in serum, clinicopathological characteristics, and the presence of (proliferating) T cells (CD8, Ki67, and FoxP3) was examined. In addition, we compared the relationships between IDO1 and IFNG gene expression and clinical parameters using RNAseq data from 144 cervical tumor samples published by The Cancer Genome Atlas (TCGA). Here, we demonstrate that patchy tumor IDO expression is associated with an increased systemic kyn/trp ratio in cervical cancer (P = 0.009), whereas marginal tumor expression at the interface with the stroma is linked to improved disease-free (DFS) (P = 0.017) and disease-specific survival (P = 0.043). The latter may be related to T cell infiltration and localized IFNγ release inducing IDO expression. Indeed, TCGA analysis of 144 cervical tumor samples revealed a strong and positive correlation between IDO1 and IFNG mRNA expression levels (P < 0.001) and a significant association with improved DFS for high IDO1 and IFNG transcript levels (P = 0.031). Unexpectedly, IDO+ tumors had higher CD8+Ki67+ T cell rates (P = 0.004). Our data thus indicate that the serum kyn/trp ratio and IDO expression in primary tumor samples are not clear-cut biomarkers for prognosis and stratification of patients with early stage cervical cancer for clinical trials implementing IDO inhibitors. Rather, a marginal IDO expression pattern in the tumor dominantly predicts favorable outcome, which might be related to IFNγ release in the cervical tumor microenvironment.

Positive End-Expiratory Pressure Ventilation Induces Longitudinal Atrophy in Diaphragm Fibers.

RATIONALE: Diaphragm weakness in critically ill patients prolongs ventilator dependency and duration of hospital stay and increases mortality and healthcare costs. The mechanisms underlying diaphragm weakness include cross-sectional fiber atrophy and contractile protein dysfunction, but whether additional mechanisms are at play is unknown. OBJECTIVES: To test the hypothesis that mechanical ventilation with positive end-expiratory pressure (PEEP) induces longitudinal atrophy by displacing the diaphragm in the caudal direction and reducing the length of fibers. METHODS: We studied structure and function of diaphragm fibers of mechanically ventilated critically ill patients and mechanically ventilated rats with normal and increased titin compliance. MEASUREMENTS AND MAIN RESULTS: PEEP causes a caudal movement of the diaphragm, both in critically ill patients and in rats, and this caudal movement reduces fiber length. Diaphragm fibers of 18-hour mechanically ventilated rats (PEEP of 2.5 cm H2O) adapt to the reduced length by absorbing serially linked sarcomeres, the smallest contractile units in muscle (i.e., longitudinal atrophy). Increasing the compliance of titin molecules reduces longitudinal atrophy. CONCLUSIONS: Mechanical ventilation with PEEP results in longitudinal atrophy of diaphragm fibers, a response that is modulated by the elasticity of the giant sarcomeric protein titin. We postulate that longitudinal atrophy, in concert with the aforementioned cross-sectional atrophy, hampers spontaneous breathing trials in critically ill patients: during these efforts, end-expiratory lung volume is reduced, and the shortened diaphragm fibers are stretched to excessive sarcomere lengths. At these lengths, muscle fibers generate less force, and diaphragm weakness ensues.

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment.

BACKGROUND: Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells. METHODS: We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models. RESULTS: Ex vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma. CONCLUSIONS: Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

α-B Crystallin Reverses High Diastolic Stiffness of Failing Human Cardiomyocytes.

BACKGROUND: Cardiomyocytes with a less distensible titin and interstitial collagen contribute to the high diastolic stiffness of failing myocardium. Their relative contributions and mechanisms underlying loss of titin distensibility were assessed in failing human hearts. METHODS AND RESULTS: Left ventricular tissue was procured in patients with aortic stenosis (AS, n=9) and dilated cardiomyopathy (DCM, n=6). Explanted donor hearts (n=8) served as controls. Stretches were performed in myocardial strips, and an extraction protocol differentiated between passive tension (Fpassive) attributable to cardiomyocytes or to collagen. Fpassive-cardiomyocytes was higher in AS and DCM at shorter muscle lengths, whereas Fpassive-collagen was higher in AS at longer muscle lengths and in DCM at shorter and longer muscle lengths. Cardiomyocytes were stretched to investigate titin distensibility. Cardiomyocytes were incubated with alkaline phosphatase, subsequently reassessed after a period of prestretch and finally treated with the heat shock protein α-B crystallin. Alkaline phosphatase shifted the Fpassive-sarcomere length relation upward only in donor. Prestretch shifted the Fpassive-sarcomere length relation further upward in donor and upward in AS and DCM. α-B crystallin shifted the Fpassive-sarcomere length relation downward to baseline in donor and to lower than baseline in AS and DCM. In failing myocardium, confocal laser microscopy revealed α-B crystallin in subsarcolemmal aggresomes. CONCLUSIONS: High cardiomyocyte stiffness contributed to stiffness of failing human myocardium because of reduced titin distensibility. The latter resulted from an absent stiffness-lowering effect of baseline phosphorylation and from titin aggregation. High cardiomyocyte stiffness was corrected by α-B crystallin probably through relief of titin aggregation.

Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors.

PSMB5 mutations and upregulation of the ß5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.