FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
Green tea reduces body fat via upregulation of neprilysin
Muenzner, M., Tappenbeck(*), N., Gembardt(*), F., Rülke, R., Furkert, J., Melzig(*), M. F., Siems, W. E., Brockmann(*), G. A.; Walther(*), T.
Int J Obes (Lond), 40:1850-1855
(2016)

Tags: Biochemical Neurobiology (Siems)

Abstract: BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Insight into the Modification of Polymeric Micellar and Liposomal Nanocarriers by Fluorescein-Labeled Lipids and Uptake-Mediating Lipopeptides
Draffehn(*), S., Eichhorst, J., Wiesner, B.; Kumke(*), M. U.
Langmuir, 32:6928-6939
(2016)

Tags: Cellular Imaging (Wiesner)

Abstract: Encapsulation of diagnostic and therapeutic compounds in transporters improves their delivery to the point of need. An even more efficient treatment of diseases can be achieved using carriers with targeting or protecting moieties. In the present work, we investigated micellar and liposomal nanocarriers modified with fluorescein, peptides, and polymers that are covalently bound to fatty acids or phospholipids to ensure a self-driven incorporation into the micelles or liposomes. First, we characterized the photophysics of the fluorescent probes in the absence and in the presence of nanocarriers. Changes in the fluorescence decay time, quantum yield, and intensity of a fluorescein-labeled fatty acid (fluorescein-labeled palmitic acid [fPA]) and a fluorescein-labeled lipopeptide (P2fA2) were found. By exploiting these changes, we investigated a lipopeptide (P2A2 as an uptake-mediating unit) in combination with different nanocarriers (micelles and liposomes) and determined the corresponding association constant Kass values, which were found to be very high. In addition, the mobility of fPA was exploited using fluorescence correlation spectroscopy (FCS) and fluorescence depolarization (FD) experiments to characterize the nanocarriers. Cellular uptake experiments with mouse brain endothelial cells provided information on the uptake behavior of liposomes modified by uptake-mediating P2A2 and revealed differences in the uptake behavior between pH-sensitive and pH-insensitive liposomes.

Lipid dynamics in boar sperm studied by advanced fluorescence imaging techniques
Schröter(*), F., Jakop(*), U., Teichmann, A., Haralampiev(*), I., Tannert(*), A., Wiesner, B., Müller(*), P.; Müller(*), K.
European biophysics journal : EBJ, 45:149-163
(2016)

Tags: Cellular Imaging (Wiesner)

Abstract: The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes
Hu(*), H., Haas(*), S. A., Chelly(*), J., Van Esch(*), H., Raynaud(*), M., de Brouwer(*), A. P., Weinert, S., Froyen(*), G., Frints(*), S. G., Laumonnier, F., Zemojtel(*), T., Love(*), M. I., Richard(*), H., Emde(*), A. K., Bienek(*), M., Jensen(*), C., Hambrock(*), M., Fischer(*), U., Langnick(*), C., Feldkamp(*), M., Wissink-Lindhout(*), W., Lebrun(*), N., Castelnau(*), L., Rucci(*), J., Montjean(*), R., Dorseuil(*), O., Billuart(*), P., Stuhlmann, T., Shaw(*), M., Corbett(*), M. A., Gardner(*), A., Willis-Owen(*), S., Tan(*), C., Friend(*), K. L., Belet(*), S., van Roozendaal(*), K. E., Jimenez-Pocquet(*), M., Moizard(*), M. P., Ronce(*), N., Sun(*), R., O'Keeffe(*), S., Chenna(*), R., van Bommel(*), A., Goke(*), J., Hackett(*), A., Field(*), M., Christie(*), L., Boyle(*), J., Haan(*), E., Nelson(*), J., Turner(*), G., Baynam(*), G., Gillessen-Kaesbach(*), G., Müller, U., Steinberger(*), D., Budny(*), B., Badura-Stronka(*), M., Latos-Bielenska(*), A., Ousager(*), L. B., Wieacker(*), P., Rodriguez Criado(*), G., Bondeson(*), M. L., Anneren(*), G., Dufke(*), A., Cohen(*), M., Van Maldergem(*), L., Vincent-Delorme(*), C., Echenne(*), B., Simon-Bouy(*), B., Kleefstra(*), T., Willemsen(*), M., Fryns(*), J. P., Devriendt(*), K., Ullmann(*), R., Vingron(*), M., Wrogemann(*), K., Wienker(*), T. F., Tzschach(*), A., van Bokhoven(*), H., Gecz(*), J., Jentsch, T. J., Chen(*), W., Ropers(*), H. H.; Kalscheuer(*), V. M.
Molecular psychiatry, 21:133-148
(2016)

Tags: Physiology and Pathology of Ion Transport (Jentsch

Abstract: X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females
Palmer(*), E. E., Stuhlmann, T., Weinert, S., Haan(*), E., Van Esch(*), H., Holvoet(*), M., Boyle(*), J., Leffler(*), M., Raynaud(*), M., Moraine(*), C., van Bokhoven(*), H., Kleefstra(*), T., Kahrizi(*), K., Najmabadi(*), H., Ropers(*), H. H., Delgado(*), M. R., Sirsi(*), D., Golla(*), S., Sommer(*), A., Pietryga(*), M. P., Chung(*), W. K., Wynn(*), J., Rohena(*), L., Bernardo(*), E., Hamlin(*), D., Faux(*), B. M., Grange(*), D. K., Manwaring(*), L., Tolmie(*), J., Joss(*), S., Cobben(*), J. M., Duijkers(*), F. A., Goehringer(*), J. M., Challman(*), T. D., Hennig(*), F., Fischer(*), U., Grimme(*), A., Suckow(*), V., Musante(*), L., Nicholl(*), J., Shaw(*), M., Lodh(*), S. P., Niu(*), Z., Rosenfeld(*), J. A., Stankiewicz(*), P., Jentsch, T. J., Gecz(*), J., Field(*), M.; Kalscheuer(*), V. M.
Molecular psychiatry,
(2016)

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.Molecular Psychiatry advance online publication, 23 August 2016; doi:10.1038/mp.2016.135.

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling
Böhme(*), M. A., Beis(*), C., Reddy-Alla(*), S., Reynolds(*), E., Mampell(*), M. M., Grasskamp, A. T., Lutzkendorf(*), J., Bergeron(*), D. D., Driller(*), J. H., Babikir(*), H., Göttfert(*), F., Robinson(*), I. M., O'Kane(*), C. J., Hell(*), S. W., Wahl(*), M. C., Stelzl(*), U., Loll(*), B., Walter, A. M.; Sigrist(*), S. J.
Nat Neurosci, 19:1311-1320
(2016)

Tags: Molecular and Theoretical Neuroscience (Walter)

Abstract: Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca(2+) channel, but molecular mechanisms controlling this are unknown. Here we found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins Syd-1 and Liprin-alpha, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca(2+) channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13A(null) mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. We identified isoform-specific Unc13-AZ scaffold interactions regulating SV-Ca(2+)-channel topology whose developmental tightening optimizes synaptic transmission.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses
Grauel(*), M. K., Maglione, M., Reddy-Alla(*), S., Willmes(*), C. G., Brockmann(*), M. M., Trimbuch(*), T., Rosenmund(*), T., Pangalos(*), M., Vardar(*), G., Stumpf(*), A., Walter, A. M., Rost(*), B. R., Eickholt(*), B. J., Haucke, V., Schmitz(*), D., Sigrist(*), S. J.; Rosenmund(*), C.
Proc Natl Acad Sci U S A, 113:11615-11620
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Molecular and Theoretical Neuroscience (Walter)

Abstract: The tight spatial coupling of synaptic vesicles and voltage-gated Ca2+ channels (CaVs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca2+ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2-deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in CaV2.1 clustering at AZs, which likely alters Ca2+ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.

5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin inhibitors
Robertson(*), M. J., Horatscheck, A., Sauer, S., von Kleist(*), L., Baker, J. R., Stahlschmidt, W., Nazare, M., Whiting(*), A., Chau(*), N., Robinson(*), P. J., Haucke, V.; McCluskey(*), A.
Org Biomol Chem, 14:11266-11278
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Medicinal Chemistry (Nazare)

Abstract: The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop(R) 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 approximately 20 muM) then phenyl (25, IC50 approximately 7.1 muM) and 1-napthyl sulfonamide (26, Pitstop(R) 2 compound, IC50 approximately 1.9 muM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 approximately 1.4, 1.6 and 1.8 muM respectively) and nine others with IC50 < 10 muM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop(R) 2 family binding, not a loss of binding, within the Pistop(R) groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.

A phosphoinositide conversion mechanism for exit from endosomes
Ketel, K., Krauss, M., Nicot(*), A. S., Puchkov, D., Wieffer(*), M., Müller(*), R., Subramanian(*), D., Schultz(*), C., Laporte(*), J.; Haucke, V.
Nature, 529:408-412
(2016)

Tags: Molecular Pharmacology and Cell Biology (Haucke), Cellular Imaging (Wiesner/Puchkov)

Abstract: Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2alpha (PI4K2alpha)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6
Boschert(*), V., Frisch(*), C., Back(*), J. W., van Pee(*), K., Weidauer(*), S. E., Muth(*), E. M., Schmieder, P., Beerbaum, M., Knappik(*), A., Timmerman(*), P.; Mueller(*), T. D.
Open biology, 6
(2016)

Tags: Solution NMR (Schmieder)

Abstract: The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis.

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