Poststroke dendritic arbor regrowth requires the actin nucleator Cobl.

Ischemic stroke is a major cause of death and long-term disability. We demonstrate that middle cerebral artery occlusion (MCAO) in mice leads to a strong decline in dendritic arborization of penumbral neurons. These defects were subsequently repaired by an ipsilateral recovery process requiring the actin nucleator Cobl. Ischemic stroke and excitotoxicity, caused by calpain-mediated proteolysis, significantly reduced Cobl levels. In an apparently unique manner among excitotoxicity-affected proteins, this Cobl decline was rapidly restored by increased mRNA expression and Cobl then played a pivotal role in poststroke dendritic arbor repair in peri-infarct areas. In Cobl knockout (KO) mice, the dendritic repair window determined to span day 2 to 4 poststroke in wild-type (WT) strikingly passed without any dendritic regrowth. Instead, Cobl KO penumbral neurons of the primary motor cortex continued to show the dendritic impairments caused by stroke. Our results thereby highlight a powerful poststroke recovery process and identified causal molecular mechanisms critical during poststroke repair.

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRß subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

Syndapin I Loss-of-Function in Mice Leads to Schizophrenia-Like Symptoms.

Schizophrenia is associated with cognitive and behavioral dysfunctions thought to reflect imbalances in neurotransmission systems. Recent screenings suggested that lack of (functional) syndapin I (PACSIN1) may be linked to schizophrenia. We therefore studied syndapin I KO mice to address the suggested causal relationship to schizophrenia and to analyze associated molecular, cellular, and neurophysiological defects. Syndapin I knockout (KO) mice developed schizophrenia-related behaviors, such as hyperactivity, reduced anxiety, reduced response to social novelty, and an exaggerated novel object response and exhibited defects in dendritic arborization in the cortex. Neuromorphogenic deficits were also observed for a schizophrenia-associated syndapin I mutant in cultured neurons and coincided with a lack of syndapin I-mediated membrane recruitment of cytoskeletal effectors. Syndapin I KO furthermore caused glutamatergic hypofunctions. Syndapin I regulated both AMPAR and NMDAR availabilities at synapses during basal synaptic activity and during synaptic plasticity-particularly striking were a complete lack of long-term potentiation and defects in long-term depression in syndapin I KO mice. These synaptic plasticity defects coincided with alterations of postsynaptic actin dynamics, synaptic GluA1 clustering, and GluA1 mobility. Both GluA1 and GluA2 were not appropriately internalized. Summarized, syndapin I KO led to schizophrenia-like behavior, and our analyses uncovered associated molecular and cellular mechanisms.

Loss of retinal ganglion cells in a new genetic mouse model for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is one of the most common causes for blindness worldwide. Although an elevated intraocular pressure (IOP) is the main risk factor, the exact pathology remained indistinguishable. Therefore, it is necessary to have appropriate models to investigate these mechanisms. Here, we analysed a transgenic glaucoma mouse model (ßB1-CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in ßB1-CTGF and wildtype mice at 5, 10 and 15 weeks of age. At 5 and 10 weeks, the IOP in both groups were comparable (P > 0.05). After 15 weeks, a significant elevated IOP was measured in ßB1-CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a- and b-wave amplitudes were significantly decreased in ßB1-CTGF retinae (both P < 0.01). Significantly fewer Brn-3a+ retinal ganglion cells (RGCs) were observed in the ßB1-CTGF group on flatmounts (P = 0.02), cross-sections (P < 0.001) and also via quantitative real-time PCR (P = 0.02). Additionally, significantly more cleaved caspase 3+ RGCs were seen in the ßB1-CTGF group (P = 0.002). Furthermore, a decrease in recoverin+ cells was observable in the ßB1-CTGF animals (P = 0.004). Accordingly, a significant down-regulation of Recoverin mRNA levels were noted (P < 0.001). Gfap expression, on the other hand, was higher in ßB1-CTGF retinae (P = 0.023). Additionally, more glutamine synthetase signal was noted (P = 0.04). Although no alterations were observed regarding photoreceptors via immunohistology, a significant decrease of Rhodopsin (P = 0.003) and Opsin mRNA (P = 0.03) was noted. We therefore assume that the ßB1-CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG.

Reward expectation facilitates context learning and attentional guidance in visual search.

Modulations of visual attention due to expectation of reward were frequently reported in recent years. Recent studies revealed that reward can modulate the implicit learning of repeated context configurations (e.g., Tseng & Lleras, 2013). We investigated the influence of reward expectations on context learning by associating colors to different reward magnitudes. Participants searched through contexts consisting of spatially distributed L-shaped distractors and a T-shaped target, with half of these objects appearing in a color associated with low, medium, or high reward. Half of these context configurations were repeatedly presented in every experimental block, whereas the other half was generated newly for every trial. Results showed an earlier and more pronounced contextual cueing effect in contexts associated with high reward compared with low reward contexts. This was visible as faster decline of response times to targets in repeated contexts associated with high reward compared with repeated low reward and novel contexts, and was reflected in the eye movement pattern as shorter distance of the first fixation to the target location. These results suggest that expectation of high reward magnitude facilitates subsequent learning of repeated context configurations. High reward also increases the efficiency of attention guidance toward the target location.

HSP27 immunization reinforces AII amacrine cell and synapse damage induced by S100 in an autoimmune glaucoma model.

Previous studies have revealed a loss of retinal ganglion cells (RGCs) and optic nerve fibers after immunization with the S100B protein. Addition of heat shock protein 27 (HSP27) also leads to a decrease of RGCs. Our present aim has been to analyze various retinal cell types after immunization with S100B or S100B + HSP27 (S100 + HSP). After 28 days, retinas were processed for immunohistology and Western blot. RGCs, immunostained for NeuN, were significantly decreased in the S100 and the S100 + HSP groups. Significantly fewer ChAT+ cells were noted in both groups, whereas parvalbumin+ cells were only affected in the S100 + HSP group. Western blot results also revealed fewer ChAT signals in both immunized groups. No changes were noted with regard to PKCα+ rod bipolar cells, whereas a significant loss of recoverin+ cone bipolar cells was observed in both groups via immunohistology and Western blot. The presynaptic marker Bassoon and the postsynaptic marker PSD95 were significantly reduced in the S100 + HSP group. Opsin+ and rhodopsin+ photoreceptors revealed no changes in either group. Thus, the inner retinal layers are affected by immunization. However, the combination of S100 and HSP27 has a stronger additive effect on the retinal synapses and AII amacrine cells.

Proteomic analysis of glycine receptor ß subunit (GlyRß)-interacting proteins: evidence for syndapin I regulating synaptic glycine receptors.

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their ß subunits (GlyRß) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRß, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRß as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRß that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRß in vitro and colocalized with GlyRß upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRß. Deletion and point mutation analysis disclosed that SdpI binding to GlyRß is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.

Structural dynamics of the cell wall precursor lipid II in the presence and absence of the lantibiotic nisin.

Representing a physiological "Achilles' heel", the cell wall precursor lipid II (LII) is a prime target for various classes of antibiotics. Over the years LII-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying LII functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of LII, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded LII in the absence and presence of the LII-binding lantibiotic nisin. In a series of 2×4 independent, unbiased 100ns MD simulations we sampled the conformational dynamics of nine LII as well as nine LII-nisin complexes embedded in an aqueous 150mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to LII induces a reduction of LII mobility and flexibility, an outward shift of the LII pentapeptide, an inward movement of the LII disaccharide section, and an overall deeper insertion of the LII tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and LII interaction remain similar to the 1WCO LII-nisin NMR solution structure.

Let's go bananas: revisiting the endocytic BAR code.

Against the odds of membrane resistance, members of the BIN/Amphiphysin/Rvs (BAR) domain superfamily shape membranes and their activity is indispensable for a plethora of life functions. While crystal structures of different BAR dimers advanced our understanding of membrane shaping by scaffolding and hydrophobic insertion mechanisms considerably, especially life-imaging techniques and loss-of-function studies of clathrin-mediated endocytosis with its gradually increasing curvature show that the initial idea that solely BAR domain curvatures determine their functions is oversimplified. Diagonal placing, lateral lipid-binding modes, additional lipid-binding modules, tilde shapes and formation of macromolecular lattices with different modes of organisation and arrangement increase versatility. A picture emerges, in which BAR domain proteins create macromolecular platforms, that recruit and connect different binding partners and ensure the connection and coordination of the different events during the endocytic process, such as membrane invagination, coat formation, actin nucleation, vesicle size control, fission, detachment and uncoating, in time and space, and may thereby offer mechanistic explanations for how coordination, directionality and effectiveness of a complex process with several steps and key players can be achieved.

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I.

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.

Unilateral access regulation: ground state dynamics of the Pseudomonas aeruginosa outer membrane efflux duct OprM.

Acting as an efflux duct in the MexA-MexB-OprM multidrug efflux pump, OprM plays a major role in the antibiotic resistance capability of Pseudomonas aeruginosa, trafficking substrates through the outer cell membrane. Whereas the available crystal structures showed restricted OprM access on both ends, the underlying gating mechanism is not yet fully understood. To gain insight into the functional mechanism of OprM access regulation, we conducted a series of five independent, unbiased molecular dynamics simulations, computing 200 ns dynamics samples of the wild-type protein in a phospholipid membrane/150 mM NaCl water environment. On the extracellular side, OprM opens and closes freely under the simulated conditions, suggesting the absence of a gating mechanism on this side of the isolated protein. On the periplasmic side, we observe an opening of the tip regions at Val408 and to a lesser degree Asp416 located 1.5 nm further into the channel, leading to OprM end conformations being up to 3 and 1.4 times, respectively, more open than the asymmetric crystal structure. If our simulations are correct, our findings imply that periplasmic gating involves only the Asp416 region and that in vivo additional components, absent in our simulation, might be required for periplasmic gating if the observed opening trend near Asp416 is not negligible. In addition to that ,we identified in each monomer a previously unreported sodium binding site in the channel interior coordinated by Asp171 and Asp230 whose functional role remains to be investigated.

Ultrastructural freeze-fracture immunolabeling identifies plasma membrane-localized syndapin II as a crucial factor in shaping caveolae.

Membrane topology control is thought to involve peripheral membrane proteins of the F-BAR domain family including syndapins. These proteins are predestined to shape membranes by partial insertion and by imposing their curved shape onto the lipid bilayer. Direct observation of such functions on cellular membranes, however, was precluded by the difficulty to combine high-resolution imaging with visualization of membrane topology. Here, we report the ultrastructural visualization of endogenous syndapin II at the plasma membrane of NIH 3T3 cells using a combination of freeze-fracturing, immunogold labeling and transmission electron microscopy. Surprisingly, syndapin II was detected at flat and curved membrane areas. Ultrastructural colocalization with caveolin 1 identified syndapin II-positive invaginations as caveolae. Consistent with the syndapin II F-BAR domain interacting with caveolin 1, F-BAR overexpression affected caveolin 1 localization. Syndapin II knockdown did not alter caveolin 1 expression or plasma membrane recruitment. Instead, syndapin II knockdown reduced the density of caveolae and strongly increased the number of caveolin 1 molecules at flat membrane areas. Comparative immunoelectron microscopy and tilt series revealed that syndapin II was asymmetrically localized at the neck of caveolae. Double-immunogold labeling showed that the caveolae-shaping molecule PTRF/cavin 1 behaved similarly and that syndapin II and PTRF/cavin 1 colocalized. Visualization of a transiently membrane-binding F-BAR protein in direct relation to membrane topology of mammalian cells thereby revealed that syndapin II binds to both flat and curved membranes in vivo and that it plays an important role in caveolar shaping, a role that it shares with PTRF/cavin 1.

Blockage of neuromuscular glutamate receptors impairs reinnervation following nerve crush in adult mice.

Motor axons in peripheral nerves are capable of regeneration following injury. However, complete recovery of motor function is rare, particularly when reinnervation is delayed. We have previously found that glutamate receptors play a crucial role in the successful innervation of muscle during mouse development. In particular, blocking N-methyl-D-aspartate (NMDA) receptor activity delays the normal elimination of excess innervation of each neuromuscular junction. Here, we use behavioral, immunohistochemical, electrophysiological, and calcium imaging methods to test whether glutamate receptors play a similar role in the transition from polyneuronal to mono-innervation and in recovery of function following peripheral nerve injury in mature muscle.

Cytokine and Complement Response in the Glaucomatous ßB1-CTGF Mouse Model.

Glaucoma is a complex neurodegenerative disease leading to a loss of retinal ganglion cells (RGCs) and optic nerve axons. An activation of the complement system seems to contribute to cell loss in this disease. Hence, we investigated a possible initiation of the complement system and the cytokine response in the ßB1-CTGF glaucoma model. In these mice, intraocular pressure is elevated, which is the main glaucoma risk factor in patients, and RGC loss occurs at 15 weeks of age. Therefore, quantitative real-time PCR and immunohistological experiments were performed in 5-, 10-, and 15-week-old ßB1-CTGF animals and their corresponding wildtypes (WT) to analyze the expression of several complement system factors. We could show that mRNA levels of the terminal complement pathway components C3 and C5 (Hc) were upregulated at 10 weeks. In accordance, more C3+ and membrane attack complex+ cells were observed in transgenic retinae. Further, the C5a receptor anaphylatoxin receptor (C5ar) and the complement component C5a receptor 1 (C5ar1; CD88) mRNA levels were upregulated in 10- and 15-week-old ßB1-CTGF mice. Interestingly, all three activation routes of the complement system were elevated in ßB1-CTGF mice at some age. Especially C1q, as a marker of the classical pathway, was significantly increased at all investigated ages. Furthermore, mRNA expression levels of interferon-γ (Infg) were upregulated at 5 weeks, while Cxcl1 and Cxcl2 mRNA levels were upregulated at 10 and 15 weeks. The mRNA levels of the chemokines Cxcl10 were increased at all ages in ßB1-CTGF mice. These results lead to the assumption that in these transgenic mice, a complement activation mainly through the classical pathway as well as a cytokine response plays a major role in cell death.

Bleeding management in computed tomography-guided liver biopsies by biopsy tract plugging with gelatin sponge slurry.

To evaluate the safety and impact of biopsy tract plugging with gelatin sponge slurry in percutaneous liver biopsy. 300 consecutive patients (158 females, 142 males; median age, 63 years) who underwent computed tomography-guided core biopsy of the liver in coaxial technique (16/18 Gauge) with and without biopsy tract plugging were retrospectively reviewed (January 2013 to May 2018). Complications were rated according to the common criteria for adverse events (NCI-CTCAE). The study cohort was dichotomized into a plugged (71%; n = 214) and an unplugged (29%; n = 86) biopsy tract group. Biopsy tract plugging with gelatin sponge slurry was technically successful in all cases. Major bleeding events were only observed in the unplugged group (0.7%; n = 2), whereas minor bleedings (4.3%) were observed in both groups (plugged, 3.6%, n = 11; unplugged, 0.7%, n = 2). Analysis of biopsies and adverse events showed a significant association between number of needle-passes and overall (P = 0.038; odds ratio: 1.395) as well as minor bleeding events (P = 0.020; odds ratio: 1.501). No complications associated with gelatin sponge slurry were observed. Biopsy tract plugging with gelatin sponge slurry is a technically easy and safe procedure that can prevent major bleeding events following liver biopsy.

F-BAR proteins of the syndapin family shape the plasma membrane and are crucial for neuromorphogenesis.

Coordinated functions of the actin cytoskeleton and microtubules, which require careful control in time and space, are indispensable for the drastic alterations of neuronal morphology during neuromorphogenesis and neuronal network formation. Actin filament formation driven by the Arp2/3 complex and its activator neural Wiskott-Aldrich syndrome protein (N-WASP) is important for proper axon development. The underlying molecular mechanisms for targeting to and specific activation of N-WASP at the neuronal plasma membrane, however, have thus far remained elusive. We show that syndapin I is critical for proper neuromorphogenesis and hereby uses N-WASP as a cytoskeletal effector. Upon N-WASP binding, syndapins release N-WASP autoinhibition. Syndapins hereby cooperate with Cdc42 and phosphatidyl-inositol-(4,5)-bisphosphate. Syndapins furthermore specifically bind to phosphatidylserine-containing membranes via their extended F-BAR domain. Dissecting the syndapin functions actin nucleation and direct membrane binding in vivo, we demonstrate that both functions are physiologically relevant and required. Constitutive plasma membrane-targeting experiments in vivo indicate that specifically actin nucleation at the cell cortex is triggered by syndapins. Consistent with syndapins steering N-WASP as downstream effector for cortical actin nucleation, syndapin-induced neuronal arborization is N-WASP and Cdc42 dependent. The functions of syndapin-N-WASP complexes in neuromorphogenesis were revealed by loss-of-function studies. Knockdown of syndapin I leads to impaired axon development and especially phenocopies the aberrant axon branching observed upon N-WASP and Arp2/3 complex deficiency. In contrast, proper length control involves another N-WASP-binding protein, Abp1. Our data thus reveal that syndapin I is crucial for neuromorphogenesis and that different N-WASP activators ensure fine control of N-WASP activity and have distinct functions during neuronal network formation.

A real-time device for converting Doppler ultrasound audio signals into fluid flow velocity.

A Doppler signal converter has been developed to facilitate cardiovascular and exercise physiology research. This device directly converts audio signals from a clinical Doppler ultrasound imaging system into a real-time analog signal that accurately represents blood flow velocity and is easily recorded by any standard data acquisition system. This real-time flow velocity signal, when simultaneously recorded with other physiological signals of interest, permits the observation of transient flow response to experimental interventions in a manner not possible when using standard Doppler imaging devices. This converted flow velocity signal also permits a more robust and less subjective analysis of data in a fraction of the time required by previous analytic methods. This signal converter provides this capability inexpensively and requires no modification of either the imaging or data acquisition system.

Ankyrin repeat-containing N-Ank proteins shape cellular membranes.

Cells of multicellular organisms need to adopt specific morphologies. However, the molecular mechanisms bringing about membrane topology changes are far from understood-mainly because knowledge of membrane-shaping proteins that can promote local membrane curvatures is still limited. Our analyses unveiled that several members of a large, previously unrecognised protein family, which we termed N-Ank proteins, use a combination of their ankyrin repeat array and an amino (N)-terminal amphipathic helix to bind and shape membranes. Consistently, functional analyses revealed that the N-Ank protein ankycorbin (NORPEG/RAI14), which was exemplarily characterised further, plays an important, ankyrin repeat-based and N-terminal amphipathic helix-dependent role in early morphogenesis of neurons. This function furthermore required coiled coil-mediated self-assembly and manifested as ankycorbin nanodomains marked by protrusive membrane topologies. In summary, here, we unveil a class of powerful membrane shapers and thereby assign mechanistic and cell biological functions to the N-Ank protein superfamily.

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling.

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.

Impaired leg vasodilation during dynamic exercise in healthy older women.

The purpose of the present study was to test the hypothesis that leg blood flow responses during leg cycle ergometry are reduced with age in healthy non-estrogen-replaced women. Thirteen younger (20-27 yr) and thirteen older (61-71 yr) normotensive, non-endurance-trained women performed both graded and constant-load bouts of leg cycling at the same absolute exercise intensities. Leg blood flow (femoral vein thermodilution), mean arterial pressure (MAP; radial artery), mean femoral venous pressure, cardiac output (acetylene rebreathing), and blood O2 contents were measured. Leg blood flow responses at light workloads (20-40 W) were similar in younger and older women. However, at moderate workloads (50-60 W), leg blood flow responses were significantly attenuated in older women. MAP was 20-25 mmHg higher (P < 0.01) in the older women across all work intensities, and calculated leg vascular conductance (leg blood flow/estimated leg perfusion pressure) was lower (P < 0.05). Exercise-induced increases in leg arteriovenous O2 difference and O2 extraction were identical between groups (P > 0.6). Leg O2 uptake was tightly correlated with leg blood flow across all workloads in both subject groups (r2 = 0.80). These results suggest the ability of healthy older women to undergo limb vasodilation in response to submaximal exercise is impaired and that the legs are a potentially important contributor to the augmented systemic vascular resistance seen during dynamic exercise in older women.