Super-resolution microscopy reveals that Na/K-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad.

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na,K-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain.

A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales.

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols.

RNA-seq reveals altered gene expression levels in proximal tubular cell cultures compared to renal cortex but not during early glucotoxicity.

Cell cultures are often used to study physiological processes in health and disease. It is well-known that cells change their gene expression in vitro compared to in vivo, but it is rarely experimentally addressed. High glucose is a known trigger of apoptosis in proximal tubular cells (PTC).

Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na, K-ATPase control of cell adhesion, proliferation, and survival.

The ion pump Na, K-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer.

Prompt apoptotic response to high glucose in SGLT-expressing renal cells.

It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet, little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure. In the present ex vivo study of rat renal cells, we compared the apoptotic response to a moderate increase in glucose concentration.

Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.

The glomerular filtration barrier, has historically only been spatially resolved using electron microscopy due to the nanometer-scale dimensions of these structures. Recently, it was shown that the nanoscale distribution of proteins in the slit diaphragm can be resolved by fluorescence based stimulated emission depletion microscopy, in combination with optical clearing. Fluorescence microscopy has advantages over electron microscopy in terms of multiplex imaging of different epitopes, and also the amount of volumetric data that can be extracted from thicker samples.

AT-receptor response to non-saturating Ang-II concentrations is amplified by calcium channel blockers.

Background: Blockers of angiotensin II type 1 receptor (ATR) and the voltage gated calcium channel 1.2 (Ca1.2) are commonly used for treatment of hypertension.

Prevention of apoptosis averts glomerular tubular disconnection and podocyte loss in proteinuric kidney disease.

There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis.

Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.

The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited.

Spatial distribution of DARPP-32 in dendritic spines.

The phosphoprotein DARPP-32 (dopamine and cyclic adenosine 3´, 5´-monophosphate-regulated phosphoprotein, 32 kDa) is an important component in the molecular regulation of postsynaptic signaling in neostriatum. Despite the importance of this phosphoprotein, there is as yet little known about the nanoscale distribution of DARPP-32. In this study we applied superresolution stimulated emission depletion microscopy (STED) to assess the expression and distribution of DARPP-32 in striatal neurons.

A noncanonical postsynaptic transport route for a GPCR belonging to the serotonin receptor family.

Postsynaptic receptor trafficking plays an essential role in tuning neurotransmission and signal plasticity and has emerged as a potential therapeutic target in neuropsychiatric disease. Using a novel application of fluorescence recovery after photobleaching in rat hippocampal neurons, we examined transport from the soma to dendrites of seven G-protein-coupled receptors (GPCRs) implicated in mood disorders. Most GPCRs were delivered to dendrites via lateral diffusion, but one GPCR, the serotonin 1B receptor (5-HT(1B)), was delivered to the dendrites in secretory vesicles.

Binding of losartan to angiotensin AT1 receptors increases dopamine D1 receptor activation.

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro.

Nearest neighbor analysis of dopamine D1 receptors and Na(+)-K(+)-ATPases in dendritic spines dissected by STED microscopy.

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact.

Spatial distribution of Na+-K+-ATPase in dendritic spines dissected by nanoscale superresolution STED microscopy.

Background: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons.

Results: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected.

Developmental changes in frequency of the ciliary somatostatin receptor 3 protein.

Primary cilia extend from the surface of most vertebrate cells and display several signaling molecules, including the somatostatin receptor 3 (SSTR3), enabling cilia to play essential roles as chemical, osmotic and mechanical sensors. The SSTR3 is widely distributed in the adult rat brain, and also influences cell proliferation and apoptosis. To establish whether the SSTR3 is positioned to influence these developmental processes, we examined, using immunohistochemistry, the embryonic and postnatal development of SSTR3 expression in the rat hippocampal formation, and its association with newly born and mature neurons in adult rats.

Negative reciprocity between angiotensin II type 1 and dopamine D1 receptors in rat renal proximal tubule cells.

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling.

Intracellular dynamics of calcyon, a neuron-specific vesicular protein.

Calcyon is a brain-specific protein, implicated in clathrin-mediated endocytosis. In this descriptive study we show that calcyon is exclusively expressed in neurons, and localized in moving vesicles. The movement of calcyon-containing vesicles was dependent on temperature and on intact microtubules, in addition these vesicles were colocalized with a marker for endocytosed plasma membrane proteins, suggesting that calcyon vesicles follow the endocytic recycling pathway.

Effect of TNF-alpha on CD3-zeta and MHC-I in postnatal rat hippocampus.

The zeta subunit of the CD3 T-cell receptor complex and the major histocompatibility complex class 1 (MHC-I) are important not only for the immune response to antigens, they also function as signal molecules in the brain, where they play a role in the postnatal maturation process. The expression of these molecules can be regulated by cytokines. In situations associated with increased cytokine production, such as neonatal hypoxia, the hippocampus is particularly susceptible to permanent damage.

Allosteric changes of the NMDA receptor trap diffusible dopamine 1 receptors in spines.

The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors.

Locomotor effects of a D1R agonist are DARPP-32 dependent in adult but not weanling mice.

Evidence suggests that dopamine regulation of motor activity undergoes postnatal maturation. To examine the role of the dopamine 1 receptor (D1R)/dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) signaling pathway for this maturation, we studied the effects of a D1R agonist on motor activity in weanling and adult wild-type (WT) mice and mice that lack DARPP-32, a key messenger in the D1R signaling pathway. Locomotor activity was not affected by D1R activation in WT weanling mice but was significantly stimulated in WT adult mice.

Alteration of dopamine D1 receptor-mediated motor inhibition and stimulation during development in rats is associated with distinct patterns of c-fos mRNA expression in the frontal-striatal circuitry.

Dopamine D1 receptors have been implicated in various neurodevelopmental disorders, including attention-deficit/hyperactivity disorder. However, little is known about potential late maturational changes of the motor inhibitory and stimulatory role of these receptors. Here, we investigated the effects of a full and selective D1 receptor agonist, SKF-81297, on motor activity and expression of the plasticity-associated gene, c-fos, in the prefrontal cortex and striatum of juvenile and adolescent male rats.

Recruitment of renal dopamine 1 receptors requires an intact microtubulin network.

Renal dopamine1 receptor (D1R) can be recruited from intracellular compartments to the plasma membrane by D1R agonists and endogenous dopamine. This study examines the role of the cytoskeleton for renal D1R recruitment. The studies were performed in LLCPK-1 cells that have the capacity to form dopamine from L-dopa.

Sex differences in the motor inhibitory and stimulatory role of dopamine D1 receptors in rats.

We investigated sex differences in the motor responses to the full and selective dopamine D1-like receptor agonist, (+/-)-6-chloro-7,8-dihydroxyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297; 0.3, 3, and 10 mg/kg, s.c.

Quantitative detection of respiratory Chlamydia pneumoniae infection by real-time PCR.

Real-time PCR was evaluated as a quantitative diagnostic method for Chlamydia pneumoniae infection using different respiratory samples. Real-time PCR had efficiency equal to or better than that of nested touchdown PCR. This study confirmed sputum as the best sampling material to detect an ongoing C.

Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation.

Glutamate, by activating N-methyl-d-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments.