KCTD12 Auxiliary Proteins Modulate Kinetics of GABAB Receptor-Mediated Inhibition in Cholecystokinin-Containing Interneurons

Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins

Lack of Neuronal IFN-β-IFNAR Causes Lewy Body- and Parkinson's Disease-like Dementia.

Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-β (IFN-β) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-β signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-β promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-β overexpression prevented dopaminergic neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-β in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.Support to S.I.-N. was from Danish Council For Independent Research (DFF)-Medical Sciences, Alzheimer-forskningsfonden, Danish Multiple Sclerosis Society, Danish Cancer Society and Lundbeck Foundation. D.C.R. is a Wellcome Trust Principal Research Fellow.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.cell.2015.08.06

Decellularized In Vitro Capillaries for Studies of Metastatic Tendency and Selection of Treatment

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: “network” and “cluster”. Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.publishedVersionPeer reviewe

Munc13-3 Is Required for the Developmental Localization of Ca2+ Channels to Active Zones and the Nanopositioning of Cav2.1 Near Release Sensors

Summary: Spatial relationships between Cav channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Cav2.1 and Cav2.2 channels, alters the localization of Cav2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca2+-channel pharmacology in electrophysiological and two-photon Ca2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca2+ influx through Cav2.1 and Cav2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Cav2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Cav2.1 and Cav2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains

Tripartite Separation of Glomerular Cell Types and Proteomes from Reporter-Free Mice

Background The glomerulus comprises podocytes, mesangial cells, and endothelial cells, which jointly determine glomerular filtration. Understanding this intricate functional unit beyond the transcriptome requires bulk isolation of these cell types for biochemical investigations. We developed a globally applicable tripartite isolation method for murine mesangial and endothelial cells and podocytes (timMEP). Methods We separated glomerular cell types from wild-type or mT/mG mice via a novel FACS approach, and validated their purity. Cell type proteomes were compared between strains, ages, and sex. We applied timMEP to the podocyte-targeting, immunologic, THSD7A-associated, model of membranous nephropathy. Results timMEP enabled protein-biochemical analyses of podocytes, mesangial cells, and endothelial cells derived from reporter-free mice, and allowed for the characterization of podocyte, endothelial, and mesangial proteomes of individual mice. We identified marker proteins for mesangial and endothelial proteins, and outlined protein-based, potential communication networks and phosphorylation patterns. The analysis detected cell type-specific proteome differences between mouse strains and alterations depending on sex, age, and transgene. After exposure to anti-THSD7A antibodies, timMEP resolved a fine-tuned initial stress response, chiefly in podocytes, that could not be detected by bulk glomerular analyses. The combination of proteomics with super-resolution imaging revealed a specific loss of slit diaphragm, but not of other foot process proteins, unraveling a protein-based mechanism of podocyte injury in this animal model. Conclusion timMEP enables glomerular cell type-resolved investigations at the transcriptional and protein-biochemical level in health and disease, while avoiding reporter-based artifacts, paving the way toward the comprehensive and systematic characterization of glomerular cell biology

The proteasome modulates endocytosis specifically in glomerular cells to promote kidney filtration

Abstract Kidney filtration is ensured by the interaction of podocytes, endothelial and mesangial cells. Immunoglobulin accumulation at the filtration barrier is pathognomonic for glomerular injury. The mechanisms that regulate filter permeability are unknown. Here, we identify a pivotal role for the proteasome in a specific cell type. Combining genetic and inhibitor-based human, pig, mouse, and Drosophila models we demonstrate that the proteasome maintains filtration barrier integrity, with podocytes requiring the constitutive and glomerular endothelial cells the immunoproteasomal activity. Endothelial immunoproteasome deficiency as well as proteasome inhibition disrupt the filtration barrier in mice, resulting in pathologic immunoglobulin deposition. Mechanistically, we observe reduced endocytic activity, which leads to altered membrane recycling and endocytic receptor turnover. This work expands the concept of the (immuno)proteasome as a control protease orchestrating protein degradation and antigen presentation and endocytosis, providing new therapeutic targets to treat disease-associated glomerular protein accumulations

CD74 and CD44 Expression on CTCs in Cancer Patients with Brain Metastasis

Up to 40% of advance lung, melanoma and breast cancer patients suffer from brain metastases (BM) with increasing incidence. Here, we assessed whether circulating tumor cells (CTCs) in peripheral blood can serve as a disease surrogate, focusing on CD44 and CD74 expression as prognostic markers for BM. We show that a size-based microfluidic approach in combination with a semi-automated cell recognition system are well suited for CTC detection in BM patients and allow further characterization of tumor cells potentially derived from BM. CTCs were found in 50% (7/14) of breast cancer, 50% (9/18) of non-small cell lung cancer (NSCLC) and 36% (4/11) of melanoma patients. The next-generation sequencing (NGS) analysis of nine single CTCs from one breast cancer patient revealed three different CNV profile groups as well as a resistance causing ERS1 mutation. CD44 and CD74 were expressed on most CTCs and their expression was strongly correlated, whereas matched breast cancer BM tissues were much less frequently expressing CD44 and CD74 (negative in 46% and 54%, respectively). Thus, plasticity of CD44 and CD74 expression during trafficking of CTCs in the circulation might be the result of adaptation strategies