Deutsche Forschungsgemeinschaft

Cell migration underlies tissue formation, maintenance, and regeneration as well as pathological conditions such as cancer invasion. Structural and molecular determinants of both tissue environment and cell behavior define whether cells migrate individually (through amoeboid or mesenchymal modes) or collectively. Using a multiparameter tuning model, we describe how dimension, density, stiffness, and orientation of the extracellular matrix together with cell determinants-including cell-cell and cell-matrix adhesion, cytoskeletal polarity and stiffness, and pericellular proteolysis-interdependently control migration mode and efficiency. Motile cells integrate variable inputs to adjust interactions among themselves and with the matrix to dictate the migration mode. The tuning model provides a matrix of parameters that control cell movement as an adaptive and interconvertible process with relevance to different physiological and pathological contexts.

Interaction with chemicals, present in drugs, food, environments, and consumer goods, is an integral part of our everyday life. However, depending on the amount and duration, such interactions can also result in adverse effects. With the increase in computational methods, the in silico methods can offer significant benefits to both regulatory needs and requirements for risk assessments and the pharmaceutical industry to assess the safety profile of a chemical. Here, we present ProTox 3.0, which incorporates molecular similarity and machine-learning models for the prediction of 61 toxicity endpoints such as acute toxicity, organ toxicity, clinical toxicity, molecular-initiating events (MOE), adverse outcomes (Tox21) pathways, several other toxicological endpoints and toxicity off-targets. All the ProTox 3.0 models are validated on independent external sets and have shown strong performance. ProTox envisages itself as a complete, freely available computational platform for in silico toxicity prediction for toxicologists, regulatory agencies, computational chemists, and medicinal chemists. The ProTox 3.0 webserver is free and open to all users, and there is no login requirement and can be accessed via https://tox.charite.de. The web server takes a 2D chemical structure as input and reports the toxicological profile of the compound for each endpoint with a confidence score and overall toxicity radar plot and network plot.

The Paleocene-Eocene thermal maximum (PETM) has been attributed to the rapid release of approximately 2000 x 10(9) metric tons of carbon in the form of methane. In theory, oxidation and ocean absorption of this carbon should have lowered deep-sea pH, thereby triggering a rapid (<10,000-year) shoaling of the calcite compensation depth (CCD), followed by gradual recovery. Here we present geochemical data from five new South Atlantic deep-sea sections that constrain the timing and extent of massive sea-floor carbonate dissolution coincident with the PETM. The sections, from between 2.7 and 4.8 kilometers water depth, are marked by a prominent clay layer, the character of which indicates that the CCD shoaled rapidly (<10,000 years) by more than 2 kilometers and recovered gradually (>100,000 years). These findings indicate that a large mass of carbon (>>2000 x 10(9) metric tons of carbon) dissolved in the ocean at the Paleocene-Eocene boundary and that permanent sequestration of this carbon occurred through silicate weathering feedback.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

Characterizing the equilibrium ensemble of folding pathways, including their relative probability, is one of the major challenges in protein folding theory today. Although this information is in principle accessible via all-atom molecular dynamics simulations, it is difficult to compute in practice because protein folding is a rare event and the affordable simulation length is typically not sufficient to observe an appreciable number of folding events, unless very simplified protein models are used. Here we present an approach that allows for the reconstruction of the full ensemble of folding pathways from simulations that are much shorter than the folding time. This approach can be applied to all-atom protein simulations in explicit solvent. It does not use a predefined reaction coordinate but is based on partitioning the state space into small conformational states and constructing a Markov model between them. A theory is presented that allows for the extraction of the full ensemble of transition pathways from the unfolded to the folded configurations. The approach is applied to the folding of a PinWW domain in explicit solvent where the folding time is two orders of magnitude larger than the length of individual simulations. The results are in good agreement with kinetic experimental data and give detailed insights about the nature of the folding process which is shown to be surprisingly complex and parallel. The analysis reveals the existence of misfolded trap states outside the network of efficient folding intermediates that significantly reduce the folding speed.

Abstract Starting from continuum mechanics principles, finite element incremental formulations for non‐linear static and dynamic analysis are reviewed and derived. The aim in this paper is a consistent summary, comparison, and evaluation of the formulations which have been implemented in the search for the most effective procedure. The general formulations include large displacements, large strains and material non‐linearities. For specific static and dynamic analyses in this paper, elastic, hyperelastic (rubber‐like) and hypoelastic elastic‐plastic materials are considered. The numerical solution of the continuum mechanics equations is achieved using isoparametric finite element discretization. The specific matrices which need be calculated in the formulations are presented and discussed. To demonstrate the applicability and the important differences in the formulations, the solution of static and dynamic problems involving large displacements and large strains are presented.

We investigated the propagation of femtosecond laser pulses through a metamaterial that has a negative index of refraction for wavelengths around 1.5 micrometers. From the interference fringes of a Michelson interferometer with and without the sample, we directly inferred the phase time delay. From the pulse-envelope shift, we determined the group time delay. In a spectral region, phase and group velocity are negative simultaneously. This means that both the carrier wave and the pulse envelope peak of the output pulse appear at the rear side of the sample before their input pulse counterparts have entered the front side of the sample.

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100-2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.

Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)-mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy.

Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered beta-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins.

In this study microglial cells isolated from brain cell cultures of newborn mice were characterized and investigated for morphology, their responses to growth factors and their functional properties. The microglial cells were phagocytic, contained nonspecific esterase activity and expressed Fc (IgG1/2b) and type-3 complement receptors. Scanning electron microscopy revealed that in analogy to brain tissue two types of microglial cells are present in the cultures: the ameboid and the ramified type which both display similar appearance by transmission electron microscopy. Interleukin 3 and the granulocyte-macrophage colony-stimulating factor were potent growth factors for the cultured microglial cells. The cells were negative for class II antigens (Ia) of the major histocompatibility antigen complex. However, upon treatment with interferon-gamma (IFN-gamma) microglial cells became Ia+ and functioned as antigen-presenting cells when tested on ovalbumin-specific Ia-restricted helper T cells. Furthermore, microglial cells exposed to IFN-gamma and endotoxin developed tumor cell cytotoxicity and produced tumor necrosis factor alpha. Taken together, microglial cells share the characteristics of cells of the macrophage lineage.

We aimed to determine the frequency and time course of the enlargement of ischemic cerebral lesions following human stroke and to study the effect of the state of perfusion on lesion enlargement. Acute lesion volumes were measured on diffusion-weighted magnetic resonance images and compared with lesion volumes measured on T2-weighted images at 7 days or later. Forty-four measurements were performed between 2 and 53 hours after stroke onset in 28 patients. Thirteen patients also had magnetic resonance perfusion imaging performed. In 12 (43%) of 28 patients the initial lesion volume increased by 20% or more. The number of studies showing enlargement of the ischemic lesion volume ranged from 12 (43%) of 28 at or after 2 hours to 10 (38%) of 26 at or after 6 hours, 5 (33%) of 15 at or after 24 hours, and 2 (33%) of 6 at or after 48 hours. In 7 of the 10 patients in whom the hypoperfusion volume acutely exceeded the volume of the abnormality on diffusion-weighted images, lesion volume increased by 20% or more. This study provided evidence that substantial enlargement of human cerebral ischemic lesion volumes can occur beyond the first 6, 12, or 24 hours after onset. A mismatch acutely between the region of hypoperfusion (larger) and the region of diffusion abnormality (smaller) may be predictive of ischemic lesion enlargement.

We used electron cryotomography to study the molecular arrangement of large respiratory chain complexes in mitochondria from bovine heart, potato, and three types of fungi. Long rows of ATP synthase dimers were observed in intact mitochondria and cristae membrane fragments of all species that were examined. The dimer rows were found exclusively on tightly curved cristae edges. The distance between dimers along the rows varied, but within the dimer the distance between F(1) heads was constant. The angle between monomers in the dimer was 70° or above. Complex I appeared as L-shaped densities in tomograms of reconstituted proteoliposomes. Similar densities were observed in flat membrane regions of mitochondrial membranes from all species except Saccharomyces cerevisiae and identified as complex I by quantum-dot labeling. The arrangement of respiratory chain proton pumps on flat cristae membranes and ATP synthase dimer rows along cristae edges was conserved in all species investigated. We propose that the supramolecular organization of respiratory chain complexes as proton sources and ATP synthase rows as proton sinks in the mitochondrial cristae ensures optimal conditions for efficient ATP synthesis.

G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β(1)-adrenergic receptor (β(1)AR), the β(2)-adrenergic receptor (β(2)AR), and the γ-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β(1)-/β(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of β(1)-/β(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.

Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud-like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.

BACKGROUND: Corticotropin-independent Cushing's syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood. METHODS: We performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro. RESULTS: Exome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushing's syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased. CONCLUSIONS: Genetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.

Abstract From cultures of Streptomyces viridochromogenes a new antibiotic, phosphinothricylalanyl‐alanine, has been isolated. The new amino acid phosphinothricin is 2‐amino‐4‐methyl‐phosphino‐butyric acid, its structure is confirmed by synthesis. The tripeptide is highly active against Gram ‐positive and Gram ‐negative bacteria and against the fungus Botrytis cinerea . Phosphinothricin is an active glutamine synthetase inhibitor.

α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. α-Synuclein (α-syn) phosphorylation at serine 129 is characteristic of Parkinson disease (PD) and related α-synulceinopathies. However, whether phosphorylation promotes or inhibits α-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical for elucidating the role of α-syn in the pathogenesis of PD and for development of therapeutic strategies for PD. To better understand the structural and molecular consequences of Ser-129 phosphorylation, we compared the biochemical, structural, and membrane binding properties of wild type α-syn to those of the phosphorylation mimics (S129E, S129D) as well as of in vitro phosphorylated α-syn using a battery of biophysical techniques. Our results demonstrate that phosphorylation at Ser-129 increases the conformational flexibility of α-syn and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation. In addition, we show that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. Our findings have significant implications for current strategies to elucidate the role of phosphorylation in modulating protein structure and function in health and disease and provide novel insight into the underlying mechanisms that govern α-syn aggregation and toxicity in PD and related α-synulceinopathies. Mounting evidence from pathologic, genetic, animal model, biochemical, and biophysical studies support the hypothesis that α-synuclein (α-syn) 3The abbreviations used are: α-syn, α-synuclein; PD, Parkinson disease; SEC, size exclusion chromatography; ThT, thioflavin T; TEM, transmission electron microscopy; MTSL, 1-oxy-2, 2, 5, 5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate; CK, casein kinase; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt); MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; LB, Lewy body; WT, wild type; HPLC, high performance liquid chromatography; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline. plays a central role in the pathogenesis of Parkinson disease (PD) and several other neurodegenerative diseases, including Alzheimer disease, multiple system atrophy, dementia with Lewy bodies, Down syndrome, and neurodegeneration with brain iron accumulation, collectively referred to as “synucleinopathies” (1Trojanowski J.Q. Lee V.M. Ann. N. Y. Acad. Sci. 2003; 991: 107-110Crossref PubMed Scopus (130) Google Scholar). Although the exact function of α-syn remains poorly understood, it is thought to play a role in regulating dopamine neurotransmission (2Abeliovich A. Schmitz Y. Farinas I. Choi-Lundberg D. Ho W.H. Castillo P.E. Shinsky N. Verdugo J.M. Armanini M. Ryan A. Hynes M. Phillips H. Sulzer D. Rosenthal A. Neuron. 2000; 25: 239-252Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar), vesicular trafficking (3Cooper A.A. Gitler A.D. Cashikar A. Haynes C.M. Hill K.J. Bhullar B. Liu K. Xu K. Strathearn K.E. Liu F. Cao S. Caldwell K.A. Caldwell G.A. Marsischky G. Kolodner R.D. Labaer J. Rochet J.C. Bonini N.M. Lindquist S. Science. 2006; 313: 324-328Crossref PubMed Scopus (1080) Google Scholar, 4Outeiro T.F. Lindquist S. Science. 2003; 302: 1772-1775Crossref PubMed Scopus (624) Google Scholar), and modulating synaptic function and plasticity (5Kahle P.J. Neumann M. Ozmen L. Haass C. Ann. N. Y. Acad. Sci. 2000; 920: 33-41Crossref PubMed Scopus (83) Google Scholar, 6George J.M. Jin H. Woods W.S. Clayton D.F. Neuron. 1995; 15: 361-372Abstract Full Text PDF PubMed Scopus (730) Google Scholar). Increasing evidence suggests that phosphorylation may be an important regulator of α-syn oligomerization, fibrillogenesis, Lewy body (LB) formation, and neurotoxicity in vivo (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar). Immunohistochemical and biochemical studies suggest that the majority of α-syn within LBs from patients with PD and related synucleinopathies (8Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar, 9Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, 10Kahle P.J. Neumann M. Ozmen L. Muller V. Jacobsen H. Spooren W. Fuss B. Mallon B. Macklin W.B. Fujiwara H. Hasegawa M. Iwatsubo T. Kretzschmar H.A. Haass C. EMBO Rep. 2002; 3: 583-588Crossref PubMed Scopus (264) Google Scholar, 11Takahashi M. Kanuka H. Fujiwara H. Koyama A. Hasegawa M. Miura M. Iwatsubo T. Neurosci. Lett. 2003; 336: 155-158Crossref PubMed Scopus (116) Google Scholar, 12Hasegawa M. Fujiwara H. Nonaka T. Wakabayashi K. Takahashi H. Lee V.M. Trojanowski J.Q. Mann D. Iwatsubo T. J. Biol. Chem. 2002; 277: 49071-49076Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) is phosphorylated at Ser-129 (Ser(P)-129). Proteinaceous inclusions formed in cellular and animal models overexpressing WT or mutant α-syn can also be stained with an antibody against Ser(P)-129. A study by Fujiwara et al. (8Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (159) Google Scholar) reported that in vitro phosphorylated α-syn (at Ser-129, using casein kinase II (CK2)) forms fibrils more readily than unmodified α-syn. Phosphorylation at Ser-129 was also reported to promote the formation of cytoplasmic inclusions in some cell culture models of synucleinopathies (13Smith W.W. Margolis R.L. Li X. Troncoso J.C. Lee M.K. Dawson V.L. Dawson T.M. Iwatsubo T. Ross C.A. J. Neurosci. 2005; 25: 5544-5552Crossref PubMed Scopus (211) Google Scholar). Together, these findings suggested that phosphorylation at Ser-129 plays an important role in modulating α-syn aggregation, LB formation, and toxicity in vivo. However, in vivo studies by Feany and co-worker (7Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (525) Google Scholar) suggest a lack of correlation between phosphorylation at Ser-129 and the level of α-synfibrillation. Overexpression of the phosphomimic S129D or coexpression of WT α-syn and G protein-coupled receptor kinase 2 (Gprk2), which phosphorylates α-syn specifically at Ser-129, in the Drosophila model of PD results in increased α-syn toxicity without an increase in the number of α-syn inclusions (compared with overexpression of WT α-syn). Interestingly, overexpression of S129A results in a significant increase (4×) in the number of inclusions and suppression of dopaminergic neuronal loss produced by expression of WT human α-syn. We considered that a rigorous examination and comparison of the biochemical and biophysical properties of phosphorylation and and WT α-syn may the as well as the molecular mechanisms by which phosphorylation at Ser-129 may α-syn aggregation and toxicity in vivo. In to the phosphorylation we and the in vitro phosphorylated of α-syn. To better understand the role of the in modulating α-syn aggregation and membrane binding we compared the structural, oligomerization, and membrane binding properties of WT α-syn to those of the phosphorylation mimics (S129E, S129D) as well as the in vitro phosphorylated of α-syn using size exclusion thioflavin and transmission electron results demonstrate that phosphorylation at Ser-129 inhibits than promotes α-syn formation in vitro. important is that the phosphorylation mimics (S129E/D) do not reproduce the effect of phosphorylation at on α-syn structure and aggregation properties in vitro. findings have significant implications for in vivo and understanding of the role of α-syn in the pathogenesis of PD and related and of and α-syn using and by used in these studies as J.C. Rochet J.C. 2003; PubMed Scopus Google Scholar). the expression and of and α-syn as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. D. G. T.M. V. J. Biol. 2002; PubMed Scopus Google Scholar). To of a a single was into α-syn at for with the mutant was 1-oxy-2, 2, 5, to with α-syn and the was as W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In Phosphorylation of or mutant α-syn was phosphorylated by at a of was in the of with the and of of α-syn. phosphorylation was at for the and the was with of the was by and the effect of phosphorylation on the aggregation of α-syn, WT α-syn was phosphorylated for at and the was with the to at with for the to the but was not studies the in at a of and formation was by using an as H.A. J. M. R.J. T. J. Biol. 2002; PubMed Scopus Google Scholar). and of from of α-syn was by the of and α-syn in at the the aggregation at the from a α-syn at at for at to was from the and on an system with a and using an was also on and stained with to the of the protein and in and on stained with or to the the membrane and the membrane was with in for at membrane was with the antibody (211) at a of or at a of or α-syn Ser-129 at a of or α-syn at a of at for the membrane was with the antibody from at for was with and with and in a at a of WT or mutant α-syn on at a of with 2 of and stained with 2 of on a electron at with a the phosphorylation and of the phosphorylated of α-syn or was using on a with a and using an or or A was and the was at In the of the HPLC, a was of and and (sodium was in which was by and was with to a of To increase the of formation, of in and at by a membrane to the at and used within and of α-syn in or was with the of to a of of was for 2 at structure of α-syn was by using a of 2 for at in a of α-syn in or and from was from and and from and from of for the A was for the molecular a of in and a in A was on the using a with of was with of and of was on of the and to and with a laser by of the to the that on the of and was with the and to the of α-syn WT or mutant α-syn in at on and was to in the and using F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and D. and D. G. of and J. 4: PubMed Scopus Google Scholar). to and using the of for the of mutant and phosphorylated by in the heteronuclear single quantum of the WT protein to the in by and for and to in WT α-syn, phosphorylated α-syn, and S129D α-syn using on WT and mutant α-syn in and as an and V. C.M. PubMed Scopus Google Scholar). as a function of the V. C.M. PubMed Scopus Google Scholar, C.M. J. Scopus Google Scholar). was at was increased from to in a with a of to the of the and to using of α-syn from the of α-syn and and the of C.M. J. Scopus Google Scholar). in from WT and phosphorylated α-syn, from the of between in the and of the for on α-syn on α-syn. or was in in with on a at a of and a of for of and the structural and between and suggest that type of a to phosphorylation at a at in the in a of in to the single by To whether a of Ser-129 by an is to the effect of phosphorylation, we in which Ser-129 was by or To provide insight into the in vivo studies with the phosphorylation the S129A was also and S129A within the human of by and the mutant in and to than as by and To whether of Ser-129 into a is to reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in we the α-syn using We that phosphorylates α-syn more than casein kinase II To phosphorylation at the mutant of α-syn was and phosphorylated in vitro to α-syn To that phosphorylation by at Ser-129, we of the antibody can be as as the of not and increases with and of elucidate the consequences of phosphorylation on the structure and of α-syn, we a of and high in of WT and α-syn, and at the and a of a high of in WT and α-syn in the in of the phosphorylation of WT α-syn by the of and Ser-129 at the in the of the the in the in which of phosphorylated and and the to and α-syn, phosphorylation at was that the of Ser-129 was at its and at its phosphorylated by phosphorylation that the of to by phosphorylation of Ser-129 in α-syn. In addition, for the in α-syn, in with studies H. E. Y. M. T. E. T. Iwatsubo T. Hasegawa M. K. PubMed Scopus Google Scholar). to the of the protein We in WT α-syn and phosphorylated α-syn. to and for WT and phosphorylated not that phosphorylation effect on the structure of α-syn. of WT, and α-syn and phosphorylated WT and α-syn and with a structure and Phosphorylation the of by of the of a the a can be that an of the of a WT α-syn, we a of the of it increased to α-syn, the and α-syn a a of be WT α-syn was its increased by to phosphorylation of α-syn at Ser-129 increased by to of to phosphorylated α-syn increased the to that phosphorylation the of by α-syn to its Phosphorylation the effect of phosphorylation on we of between a specifically and than of of an increase in D. J. Biol. PubMed Scopus Google Scholar). This effect an on the the of between the and the in H. S. 25: PubMed Scopus Google Scholar). the of α-syn was into a to provide an for the the of the the of the the or the of aggregation for α-syn W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). the for WT and phosphorylated α-syn. In WT α-syn, the of a effect to and with in with W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). In phosphorylated α-syn, the was to the of the for and for to To the of phosphorylation at Ser-129 on we in WT and α-syn. of the of WT and α-syn, heteronuclear R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and as R. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphorylation at Ser-129 not in the that of the of α-syn that to of the on the to by phosphorylation not the of into a can the effect of phosphorylation on the of α-syn, we S129D and α-syn by of α-syn was to that for the WT to the of the and and we of and for S129D and α-syn to WT protein but than that for phosphorylated α-syn of Ser-129 into or does not the that WT α-syn. Phosphorylation at Ser-129 the of of Ser-129 phosphorylation on the structure of membrane-bound α-syn, of α-syn compared with of protein A and of the protein is thought to that of the membrane-bound of the protein D. E. R. G. J. Biol. PubMed Scopus Google Scholar). the of phosphorylation, that Ser-129 phosphorylation does not to structural of of the protein In the between the and the binding D. E. R. G. J. Biol. PubMed Scopus Google Scholar, R. D. J. Biol. 2003; PubMed Scopus (345) Google Scholar, A. R.L. J. Biol. 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V. 2006; PubMed Scopus Google Scholar), and a of an effect by Ser-129 phosphorylation be on these is to a to binding to To the effect of phosphorylation at Ser-129 on we the structure of the in the and of by In we that phosphorylation does not perturb the of and its to binding to studies to the effect of phosphorylation on membrane binding by multiple in as the the effect of the on the aggregation properties of α-syn, we compared the aggregation of and S129A to that of the WT protein as a function of using the binding and In a the formed more fibrils than WT protein but these not and in other in formation between the and In we that the S129A more and forms more fibrils than the WT and the phosphorylation S129A and α-syn formed fibrils with to that of WT α-syn Phosphorylation the effect of in vitro phosphorylation on α-syn formation, WT α-syn was with in the at for which the to and formation was by and and compared with that to the in the of the We that phosphorylation inhibits α-syn formation, of as from the and studies To the and we also the of α-syn at using by the of the to the that the of α-syn in to phosphorylation remains of formation the of α-syn with with the and In we not of that can be by SEC, we the of in the Phosphorylation at Ser-129 to the of studies that phosphorylates α-syn at and Ser-129 M. J. Koyama A. S. M. Iwatsubo T. L. P.J. Haass C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To the of phosphorylation at Ser-129 to the of α-syn formation, we the effect of phosphorylation on the of mutant of α-syn. of with results in significant of α-syn to the forms of the of formed significant of the of α-syn 5, A and with the and the of the of the aggregation the of with To demonstrate that of α-syn fibrillogenesis is to phosphorylation at Ser-129, we the phosphorylated of and compared its aggregation properties to that of and WT α-syn. WT, and α-syn to aggregation at with and not fibrils and in a of the and WT α-syn high of as by the increased and the significant in of with and of by However, of in the formation of that from those formed by and WT α-syn the of in a that of α-syn is thought to be phosphorylated at Y. Iwatsubo T. Hasegawa M. Lett. PubMed Scopus Google Scholar), we to whether the forms and of α-syn and the consequences of on the aggregation of α-syn. In the of of we not significant in the of However, in the of we significant of α-syn as by TEM, ThT, and and aggregation of of α-syn was by and of aggregation, of α-syn to level as at findings suggest that of aggregation by is Phosphorylation of α-syn at Ser-129 in the pathogenesis of PD and related However, the exact mechanisms by which phosphorylation the and properties of α-syn in vivo unknown. To understand the structural underlying the effect of phosphorylation on the and properties of α-syn, we the effect of phosphorylation at Ser-129 on the conformational and properties of α-syn. Phosphorylation at Ser-129 in studies that α-syn that by and to and aggregation W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar, K. J. M. C.M. J. Chem. 2005; PubMed Scopus Google Scholar). binding and that aggregation in structure W. C. T.M. M. Acad. Sci. S. A. 2005; PubMed Scopus Google Scholar). also that high of the of α-syn, to an we the effect of phosphorylation on the of α-syn. by that phosphorylation of α-syn at Ser-129 increased the of α-syn In addition, of and that phosphorylation at Ser-129 phosphorylation the of by α-syn in may its as well as function of α-syn. Ser-129 is the of α-syn that is to its with phosphorylation at these is not to α-syn membrane Phosphorylation at Ser-129 not the binding of α-syn to it with the formation of structure as by the the of the protein in its is also by Ser-129 phosphorylation, with to the of the phosphorylation within the and not into the binding This that of Ser-129 phosphorylation on the function of α-syn, which is to be with its membrane-bound is to be by the of the on the the of Phosphorylation at and used to the structural and consequences of protein phosphorylation, a comparison between the phosphorylation mimics and the phosphorylated of the protein is Our in vitro studies demonstrate that the and S129D do not reproduce the effect of phosphorylation on the structural and aggregation properties of α-syn in vitro. of Ser-129 into or not to an of α-syn and the and of to the of that of or to or the effect of phosphorylation on the structure and of α-syn and other Together, these findings the critical of the increased of the to the of and in the structural and consequences of studies have reported that by serine is by In some of these studies it was that the N. B. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), or conformational S. Acad. Sci. S. A. PubMed Scopus Google Scholar) by the than its is for the of can that phosphorylation of α-syn may an important and regulator of α-syn aggregation and with other have important implications for of to elucidate the role of phosphorylation in modulating in vivo and the of results using and for molecular understanding of the consequences of phosphorylation may insight into the of α-syn and the mechanisms by which it to neurodegeneration in PD and related to the effect of phosphorylation on α-syn aggregation and toxicity was by Feany and co-worker (7Chen L. Feany M.B. Nat. 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We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.