Adeno-associated virus-based gene therapy treats inflammatory kidney disease in mice.

Adeno-associated virus (AAV) is a promising in vivo gene delivery platform showing advantages in delivering therapeutic molecules to difficult or undruggable cells. However, natural AAV serotypes have insufficient transduction specificity and efficiency in kidney cells. Here, we developed an evolution-directed selection protocol for renal glomeruli and identified what we believe to be a new vector termed AAV2-GEC that specifically and efficiently targets the glomerular endothelial cells (GEC) after systemic administration and maintains robust GEC tropism in healthy and diseased rodents. AAV2-GEC-mediated delivery of IdeS, a bacterial antibody-cleaving proteinase, provided sustained clearance of kidney-bound antibodies and successfully treated antiglomerular basement membrane glomerulonephritis in mice. Taken together, this study showcases the potential of AAV as a gene delivery platform for challenging cell types. The development of AAV2-GEC and its successful application in the treatment of antibody-mediated kidney disease represents a significant step forward and opens up promising avenues for kidney medicine.

Emerging Insights into Keratin 16 Expression during Metastatic Progression of Breast Cancer.

Keratins are the main identification markers of circulating tumor cells (CTCs); however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p < 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

SIPA1L2 controls trafficking and local signaling of TrkB-containing amphisomes at presynaptic terminals.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.

Systematic Affinity Purification Coupled to Mass Spectrometry Identified p62 as Part of the Cannabinoid Receptor CB2 Interactome.

The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.

Drosophila homologue of Diaphanous 1 (DIAPH1) controls the metastatic potential of colon cancer cells by regulating microtubule-dependent adhesion.

Drosophila homologue of Diaphanous 1 (DIAPH1) regulates actin polymerization and microtubule (MT) stabilization upon stimulation with lysophosphatidic acid (LPA). Recently, we showed strongly reduced lung metastasis of DIAPH1-depleted colon cancer cells but we found accumulations of DIAPH1-depleted cells in bone marrow. Here, we analyzed possible organ- or tissue-specific metastasis of DIAPH1-depleted HCT-116 cells. Our data confirmed that depletion of DIAPH1 strongly inhibited lung metastasis and revealed that, in contrast to control cells, DIAPH1-depleted cells did not form metastases in further organs. Detailed mechanistic analysis on cells that were not stimulated with LPA to activate the cytoskeleton-modulating activity of DIAPH1, revealed that even under basal conditions DIAPH1 was essential for cellular adhesion to collagen. In non-stimulated cells DIAPH1 did not control actin dynamics but, interestingly, was essential for stabilization of microtubules (MTs). Additionally, DIAPH1 controlled directed vesicle trafficking and with this, local clustering of the adhesion protein integrin-ß1 at the plasma membrane. Therefore, we conclude that under non-stimulating conditions DIAPH1 controls cellular adhesion by stabilizing MTs required for local clustering of integrin-ß1 at the plasma membrane. Thus, blockade of DIAPH1-tubulin interaction may be a promising approach to inhibit one of the earliest steps in the metastatic cascade of colon cancer.

The catalytic domain of inositol-1,4,5-trisphosphate 3-kinase-a contributes to ITPKA-induced modulation of F-actin.

Inositol-1,4,5-trisphosphate-3-kinase-A (ITPKA) has been considered as an actin bundling protein because its N-terminal actin binding domain (ABD) induces formation of linear actin bundles. Since in many cancer cell lines ITPKA is essential for formation of lamellipodia, which consist of cross-linked actin filaments, here we analyzed if full length-ITPKA may induce formation of more complex actin structures. Indeed, we found that incubation of F-actin with ITPKA resulted in formation of dense, branched actin networks. Based on our result that ITPKA does not exhibit an additional C-terminal ABD, we exclude that ITPKA cross-links actin filaments by simultaneous F-actin binding with two different ABDs. Instead, stimulated-emission-depletion-microscopy and measurement of InsP3 Kinase activity give evidence that that N-terminal ABD-homodimers of ITPKA bind to F-actin while the monomeric C-termini insert between adjacent actin filaments. Thereby, they prevent formation of thick actin bundles but induce formation of thin branched actin structures. Interestingly, when embedded in this dense actin network, InsP3 Kinase activity is doubled and the product of InsP3 Kinase activity, Ins(1,3,4,5)P4 , inhibits spontaneous actin polymerization which may reflect a local negative feedback regulation of InsP3 Kinase activity. In conclusion, we demonstrate that not only the ABD of ITPKA modulates actin dynamics but reveal that the InsP3 Kinase domain substantially contributes to this process.

Decreased bone formation and increased osteoclastogenesis cause bone loss in mucolipidosis II.

Mucolipidosis type II (MLII) is a severe multi-systemic genetic disorder caused by missorting of lysosomal proteins and the subsequent lysosomal storage of undegraded macromolecules. Although affected children develop disabling skeletal abnormalities, their pathogenesis is not understood. Here we report that MLII knock-in mice, recapitulating the human storage disease, are runted with accompanying growth plate widening, low trabecular bone mass and cortical porosity. Intralysosomal deficiency of numerous acid hydrolases results in accumulation of storage material in chondrocytes and osteoblasts, and impaired bone formation. In osteoclasts, no morphological or functional abnormalities are detected whereas osteoclastogenesis is dramatically increased in MLII mice. The high number of osteoclasts in MLII is associated with enhanced osteoblastic expression of the pro-osteoclastogenic cytokine interleukin-6, and pharmacological inhibition of bone resorption prevented the osteoporotic phenotype of MLII mice. Our findings show that progressive bone loss in MLII is due to the presence of dysfunctional osteoblasts combined with excessive osteoclastogenesis. They further underscore the importance of a deep skeletal phenotyping approach for other lysosomal diseases in which bone loss is a prominent feature.

A heterogeneous in vitro three dimensional model of tumour-stroma interactions regulating sprouting angiogenesis.

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model--a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis.