Rewiring neuronal microcircuits of the brain via spine head protrusions--a role for synaptopodin and intracellular calcium stores.

Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient

Three-dimensional topology dataset of folded radar stratigraphy in northern Greenland

We present a dataset of reconstructed three-dimensional (3D) englacial stratigraphic horizons in northern Greenland. The data cover four different regions representing key ice-dynamic settings in Greenland: (i) the onset of Petermann Glacier, (ii) a region upstream of the 79° North Glacier (Nioghalvfjerdsbræ), near the northern Greenland ice divide, (iii) the onset of the Northeast Greenland Ice Stream (NEGIS) and (iv) a 700 km wide region extending across the central ice divide over the entire northern part of central Greenland. In this paper, we promote the advantages of a 3D perspective of deformed englacial stratigraphy and explain how 3D horizons provide an improved basis for interpreting and reconstructing the ice-dynamic history. The 3D horizons are provided in various formats to allow a wide range of applications and reproducibility of results

Three-dimensional topology dataset of folded radar stratigraphy in northern Greenland

We present a dataset of reconstructed three-dimensional (3D) englacial stratigraphic horizons in northern Greenland. The data cover four different regions representing key ice-dynamic settings in Greenland: (i) the onset of Petermann Glacier, (ii) a region upstream of the 79° North Glacier (Nioghalvfjerdsbræ), near the northern Greenland ice divide, (iii) the onset of the Northeast Greenland Ice Stream (NEGIS) and (iv) a 700 km wide region extending across the central ice divide over the entire northern part of central Greenland. In this paper, we promote the advantages of a 3D perspective of deformed englacial stratigraphy and explain how 3D horizons provide an improved basis for interpreting and reconstructing the ice-dynamic history. The 3D horizons are provided in various formats to allow a wide range of applications and reproducibility of results

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

Nephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic beta-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in beta-cell function. Nephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin-transfected cells was used to study nephrin endocytosis. Nephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin-transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release. Our data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes

A human glomerular SAGE transcriptome database

Background: To facilitate in the identification of gene products important in regulating renal glomerular structure and function, we have produced an annotated transcriptome database for normal human glomeruli using the SAGE approach. Description: The database contains 22,907 unique SAGE tag sequences, with a total tag count of 48,905. For each SAGE tag, the ratio of its frequency in glomeruli relative to that in 115 non-glomerular tissues or cells, a measure of transcript enrichment in glomeruli, was calculated. A total of 133 SAGE tags representing well-characterized transcripts were enriched 10-fold or more in glomeruli compared to other tissues. Comparison of data from this study with a previous human glomerular Sau3A-anchored SAGE library reveals that 47 of the highly enriched transcripts are common to both libraries. Among these are the SAGE tags representing many podocyte-predominant transcripts like WT-1, podocin and synaptopodin. Enrichment of podocyte transcript tags SAGE library indicates that other SAGE tags observed at much higher frequencies in this glomerular compared to non-glomerular SAGE libraries are likely to be glomerulus-predominant. A higher level of mRNA expression for 19 transcripts represented by glomerulus-enriched SAGE tags was verified by RT-PCR comparing glomeruli to lung, liver and spleen. Conclusions: The database can be retrieved from, or interrogated online at http://cgap.nci.nih.gov/SAGE. The annotated database is also provided as an additional file with gene identification for 9,022, and matches to the human genome or transcript homologs in other species for 1,433 tags. It should be a useful tool for in silico mining of glomerular gene expression

IQGAP1 Interacts with Components of the Slit Diaphragm Complex in Podocytes and Is Involved in Podocyte Migration and Permeability In Vitro

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties

Metalloprotease Meprinβ in Rat Kidney: Glomerular Localization and Differential Expression in Glomerulonephritis

Meprin (EC 3.4.24.18) is an oligomeric metalloendopeptidase found in microvillar membranes of kidney proximal tubular epithelial cells. Here, we present the first report on the expression of meprinβ in rat glomerular epithelial cells and suggest a potential involvement in experimental glomerular disease. We detected meprinβ in glomeruli of immunostained rat kidney sections on the protein level and by quantitative RT-PCR of laser-capture microdissected glomeruli on the mRNA level. Using immuno-gold staining we identified the membrane of podocyte foot processes as the main site of meprinβ expression. The glomerular meprinβ expression pattern was altered in anti-Thy 1.1 and passive Heymann nephritis (PHN). In addition, the meprinβ staining pattern in the latter was reminiscent of immunostaining with the sheep anti-Fx1A antiserum, commonly used in PHN induction. Using Western blot and immunoprecipitation assays we demonstrated that meprinβ is recognized by Fx1A antiserum and may therefore represent an auto-antigen in PHN. In anti-Thy 1.1 glomerulonephritis we observed a striking redistribution of meprinβ in tubular epithelial cells from the apical to the basolateral side and the cytosol. This might point to an involvement of meprinβ in this form of glomerulonephritis

Renal Thrombotic Microangiopathy in Mice with Combined Deletion of Endocytic Recycling Regulators EHD3 and EHD4

Eps15 Homology Domain-containing 3 (EHD3), a member of the EHD protein family that regulates endocytic recycling, is the first protein reported to be specifically expressed in the glomerular endothelium in the kidney; therefore we generated Ehd3–/– mice and assessed renal development and pathology. Ehd3–/– animals showed no overt defects, and exhibited no proteinuria or glomerular pathology. However, as the expression of EHD4, a related family member, was elevated in the glomerular endothelium of Ehd3–/– mice and suggested functional compensation, we generated and analyzed Ehd3–/–; Ehd4–/– mice. These mice were smaller, possessed smaller and paler kidneys, were proteinuric and died between 3–24 weeks of age. Detailed analyses of Ehd3–/–; Ehd4–/– kidneys demonstrated thrombotic microangiopathy (TMA)-like glomerular lesions including thickening and duplication of glomerular basement membrane, endothelial swelling and loss of fenestrations. Other changes included segmental podocyte foot process effacement, mesangial interposition, and abnormal podocytic and mesangial marker expression. The glomerular lesions observed were strikingly similar to those seen in human pre-eclampsia and mouse models of reduced VEGF expression. As altered glomerular endothelial VEGFR2 expression and localization and increased apoptosis was observed in the absence of EHD3 and EHD4, we propose that EHD-mediated endocytic traffic of key surface receptors such as VEGFR2 is essential for physiological control of glomerular function. Furthermore, Ehd3–/–; Ehd4–/– mice provide a unique model to elucidate mechanisms of glomerular endothelial injury which is observed in a wide variety of human renal and extra-renal diseases