Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue.

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part III: Discovery of pre-clinical development candidate BI 186908.

Although overweight and obesity are highly prevalent conditions, options to treat them are still very limited. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, two series of pyridones and pyridazinones were evaluated. Optimization was aimed at improving DMPK properties by increasing metabolic stability and improving the safety profile by reducing inhibition of the hERG channel and reducing the potential to induce phospholipidosis. Steric shielding of a labile keto moiety with an ortho-methyl group and fine-tuning of the polarity in several parts of the molecule resulted in BI 186908 (11 g), a potent and selective MCH-R1 antagonist with favorable DMPK and CMC properties. Chronic administration of BI 186908 resulted in significant body weight reduction comparable to sibutramine in a 4 week diet-induced obesity model in rats. Based on its favorable safety profile, BI 186908 was advanced to pre-clinical development.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part I: Optimization of HTS hits towards an in vivo efficacious tool compound BI 414.

Despite recent approvals of anti-obesity drugs there is still a high therapeutic need for alternative options with higher efficacy in humans. As part of our MCH-R1 antagonist program for the treatment of obesity, a series of biphenylacetamide HTS hits was evaluated. Several issues of the initial lead structures had to be resolved, such as potency, selectivity over related GPCRs and P-gp efflux limiting brain exposure in this series. We could demonstrate that all parameters can be significantly improved by structural modifications resulting in BI 414 as a potent and orally available MCH-R1 antagonist tool compound with acceptable in vivo efficacy in an animal model of obesity.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part II: Optimization of pyridazines toward reduced phospholipidosis and hERG inhibition.

Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.

Suppression of toxic transgene expression by optimized artificial miRNAs increases AAV vector yields in HEK-293 cells.

Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in both preclinical research and therapeutic applications, making the production of high-titer AAV preparations essential. To date, most AAV-based studies use constitutive promoters (e.g., CMV, CAG), which are also active in human embryonic kidney (HEK)-293 producer cells, thus leading to the expression of the transgene already during production. Depending on the transgene's function, this might negatively impact producer cell performance and result in decreased AAV vector yields. Here, we evaluated a panel of diverse microRNA (miRNA)-based shRNA designs to identify a highly potent artificial miRNA for the transient suppression of transgenes during AAV production. Our results demonstrate that insertion of miRNA target sites into the 3' UTR of the transgene and simultaneous expression of the corresponding miRNA from the 3' UTR of conventional AAV production plasmids (rep/cap, pHelper) enabled efficient silencing of toxic transgene expression, thereby increasing AAV vector yields up to 240-fold. This strategy not only allows to maintain the traditional triple-transfection protocol, but also represents a universally applicable approach to suppress toxic transgenes, thereby boosting vector yields with so far unprecedented efficiency.

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Crystal structure of glucokinase regulatory protein.

Glucokinase (GK) plays a major role in the regulation of blood glucose homeostasis in both the liver and the pancreas. In the liver, GK is controlled by the GK regulatory protein (GKRP). GKRP in turn is activated by fructose 6-phosphate (F6P) and inactivated by fructose 1-phosphate (F1P). Disrupting the GK-GKRP complex increases the activity of GK in the cytosol and is considered an attractive concept for the regulation of blood glucose. We have determined the crystal structure of GKRP in its inactive F1P-bound form. The binding site for F1P is located deeply buried at a domain interface, and H-D exchange experiments confirmed that F1P and F6P compete for this site. The structure of the inactive GKRP-F1P complex provides a starting point for understanding the mechanism of fructose phosphate-dependent GK regulation at an atomic level.

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human.

Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies. We discovered splicing patterns like the expression of a kinase-dead isoform of IRAK1 upon T-cell activation. The immunoproteasome is up-regulated in both Treg and CD4(+) Th cells upon activation, whereas the 'standard' proteasome is up-regulated in Tregs only upon activation.

Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery.

Transgenic animals with increased or abrogated target gene expression are powerful tools for drug discovery research. Here, we developed a CRISPR-based Rosa26-LSL-dCas9-VPR mouse model for targeted induction of endogenous gene expression using different Adeno-associated virus (AAV) capsid variants for tissue-specific gRNAs delivery. To show applicability of the model, we targeted low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), either individually or together. We induced up to ninefold higher expression of hepatocellular proteins. In consequence of LDLR upregulation, plasma LDL levels almost abolished, whereas upregulation of PCSK9 led to increased plasma LDL and cholesterol levels. Strikingly, simultaneous upregulation of both LDLR and PCSK9 resulted in almost unaltered LDL levels. Additionally, we used our model to achieve expression of all α1-Antitrypsin (AAT) gene paralogues simultaneously. These results show the potential of our model as a versatile tool for optimized targeted gene expression, alone or in combination.

Deep learning-based quantification of NAFLD/NASH progression in human liver biopsies.

Non-alcoholic fatty liver disease (NAFLD) affects about 24% of the world's population. Progression of early stages of NAFLD can lead to the more advanced form non-alcoholic steatohepatitis (NASH), and ultimately to cirrhosis or liver cancer. The current gold standard for diagnosis and assessment of NAFLD/NASH is liver biopsy followed by microscopic analysis by a pathologist. The Kleiner score is frequently used for a semi-quantitative assessment of disease progression. In this scoring system the features of active injury (steatosis, inflammation, and ballooning) and a separated fibrosis score are quantified. The procedure is time consuming for pathologists, scores have limited resolution and are subject to variation. We developed an automated deep learning method that provides full reproducibility and higher resolution. The system was established with 296 human liver biopsies and tested on 171 human liver biopsies with pathologist ground truth scores. The method is inspired by the way pathologist's analyze liver biopsies. First, the biopsies are analyzed microscopically for the relevant histopathological features. Subsequently, histopathological features are aggregated to a per-biopsy score. Scores are in the identical numeric range as the pathologist's ballooning, inflammation, steatosis, and fibrosis scores, but on a continuous scale. Resulting scores followed a pathologist's ground truth (quadratic weighted Cohen's κ on the test set: for steatosis 0.66, for inflammation 0.24, for ballooning 0.43, for fibrosis 0.62, and for the NAFLD activity score (NAS) 0.52. Mean absolute errors on a test set: for steatosis 0.29, for inflammation 0.53, for ballooning 0.61, for fibrosis 0.78, and for the NAS 0.77).

A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells.

Phagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.

CD276 is an important player in macrophage recruitment into the tumor and an upstream regulator for PAI-1.

More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7-H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.

Ubiquitin as potential cerebrospinal fluid marker of Creutzfeldt-Jakob disease.

Until today, a definite diagnosis of Creutzfeldt-Jakob disease (CJD) can only be made neuropathologically. At lifetime the early and differential diagnosis is often a problem. With SELDI we analyzed cerebrospinal fluid (CSF) from 32 CJD patients, 32 patients having other dementive diseases and 31 non-demented control subjects for diagnosis-dependent protein pattern differences. In a screening set of patients, peaks that discriminate best between groups were identified. These peaks were subsequently analyzed using an independent validation set of patients. Diagnostic accuracies were compared with established markers like tau protein and 14-3-3-protein. Potential marker proteins were purified and identified by LC-MS/MS. In the validation set only one peak of 8.6 kDa out of ten in the screening set could be confirmed. This protein was identified to be ubiquitin and increased levels in CSF (but not in serum) of CJD patients were confirmed by Western blot. Ubiquitin allows the correct diagnoses of that CJD cases missed by tau protein or 14-3-3-protein. We conclude that ubiquitin is a promising additional CSF biomarker for diagnosis of CJD, especially in differential diagnostically difficult cases. The selective increase of ubiquitin in CSF of CJD patients might point to an involvement of ubiquitin in pathophysiological process.

EGFL6 is increasingly expressed in human obesity and promotes proliferation of adipose tissue-derived stromal vascular cells.

With increasing rates of obesity driving the incidence of type 2 diabetes and cardiovascular diseases to epidemic levels, understanding of the biology of adipose tissue expansion is a focus of current research. Identification and characterization of secreted proteins of the adipose tissue could provide further insights into the function of adipose tissue and might help to therapeutically influence the development of obesity and associated metabolic disorders. In the present study, we identified human epidermal growth factor-like domain multiple-6 (EGFL6) as an adipose tissue-secreted protein. EGFL6 expression in human subcutaneous adipose tissue significantly increased with obesity and decreased after weight loss. Further, expression and secretion of EGFL6 increased with in vitro differentiation of human preadipocytes, suggesting that mature adipocytes are the main source of EGFL6. Containing epidermal growth factor (EGF)-like repeats, an Arg-Gly-Asp (RGD) integrin binding motif and a mephrin, A5 protein and receptor protein-tyrosine phosphatase mu (MAM) domain, EGFL6 was suggested to be an extra-cellular matrix protein. Recombinant human EGFL6 protein mediated cell adhesion of human adipose tissue-derived stromal vascular cells (AD-SVC) in an RGD-dependent manner. FACS analyses revealed specific binding of the protein to the cell surface of AD-SVC with the binding being predominantly mediated by the EGF-like repeats. Recombinant EGFL6 enhanced proliferation of human AD-SVC as measured by MTS assay and [(14)C]-thymidine incorporation. These results indicate that human EGFL6 is a paracrine/autocrine growth factor of adipose tissue up-regulated in obesity and potentially involved in the process of adipose tissue expansion and the development of obesity.

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K.

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O-glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel-associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin-deficient mice have unraveled a critical role of endosialin in site-specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac-2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C-terminal fragment of Mac-2 BP/90K, which was shown to contain binding sites for galectin-3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac-2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up-regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac-2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss-of-function adhesion experiments of Mac-2 BP/90K-expressing tumor cells on endosialin-expressing fibroblasts revealed a repulsive outcome of the Mac-2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac-2 BP/90K on tumor cells.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Short-term intra-arterial infusion of monocyte chemoattractant protein-1 results in sustained collateral artery growth.

Monocyte chemoattractant protein-1 (MCP-1) is a stimulator of collateral artery growth and has been shown to increase collateral artery conductance in rabbits and pigs. The minimal infusion duration and the minimally effective dose of MCP-1 are currently unknown, as is the sustainability of the therapeutic effect over a longer observation period than tested before. MCP-1 was infused intra-arterially in pigs after unilateral femoral artery occlusion in different doses and infusion durations between 2 hours and 2 weeks. Two weeks after ligation, arterial conductance under maximal vasodilatation was measured. The long-term efficacy was investigated in 2 additional groups of animals after 6 weeks. Infusion with 2 microg/min of MCP-1 for 6 hours was sufficient to double arterial conductance, and arterial conductance after 6 weeks was still significantly increased.

Targeting blood-brain-barrier transcytosis - perspectives for drug delivery.

Efficient transcytosis across the blood-brain-barrier (BBB) is an important strategy for accessing drug targets within the central nervous system (CNS). Despite extensive research the number of studies reporting successful delivery of macromolecules or macromolecular complexes to the CNS has remained very low. In order to expand current research it is important to know which receptors are selective and abundant on the BBB so that novel CNS-targeting antibodies or other ligands could be developed, targeting those receptors for transcytosis. To do that, we have set up a proteomics- and transcriptomics-based workflow within the COMPACT project (Collaboration on the Optimization of Macromolecular Pharmaceutical Access to Cellular Targets) of the Innovative Medicines Initiative (IMI) of the EU. Here we summarise our overall strategy in endothelial transcytosis research, describe in detail the related challenges, and discuss future perspectives of these studies. This article is part of the Special Issue entitled "Beyond small molecules for neurological disorders".