Developmental origin of oligodendrocytes determines their function in the adult brain.

In the mouse embryonic forebrain, developmentally distinct oligodendrocyte progenitor cell populations and their progeny, oligodendrocytes, emerge from three distinct regions in a spatiotemporal gradient from ventral to dorsal. However, the functional importance of this oligodendrocyte developmental heterogeneity is unknown. Using a genetic strategy to ablate dorsally derived oligodendrocyte lineage cells (OLCs), we show here that the areas in which dorsally derived OLCs normally reside in the adult central nervous system become populated and myelinated by OLCs of ventral origin. These ectopic oligodendrocytes (eOLs) have a distinctive gene expression profile as well as subtle myelination abnormalities. The failure of eOLs to fully assume the role of the original dorsally derived cells results in locomotor and cognitive deficits in the adult animal. This study reveals the importance of developmental heterogeneity within the oligodendrocyte lineage and its importance for homeostatic brain function.

Problems and Pitfalls of Identifying Remyelination in Multiple Sclerosis.

Regenerative medicines that promote remyelination in multiple sclerosis (MS) are making the transition from laboratory to clinical trials. While animal models provide the experimental flexibility to analyze mechanisms of remyelination, here we discuss the challenges in understanding where and how remyelination occurs in MS.

The fatty acid binding protein FABP7 is required for optimal oligodendrocyte differentiation during myelination but not during remyelination.

The major constituents of the myelin sheath are lipids, which are made up of fatty acids (FAs). The hydrophilic environment inside the cells requires FAs to be bound to proteins, preventing their aggregation. Fatty acid binding proteins (FABPs) are one class of proteins known to bind FAs in a cell. Given the crucial role of FAs for myelin sheath formation we investigated the role of FABP7, the major isoform expressed in oligodendrocyte progenitor cells (OPCs), in developmental myelination and remyelination. Here, we show that the knockdown of Fabp7 resulted in a reduction of OPC differentiation in vitro. Consistent with this result, a delay in developmental myelination was observed in Fabp7 knockout animals. This delay was transient with full myelination being established before adulthood. FABP7 was dispensable for remyelination, as the knockout of Fapb7 did not alter remyelination efficiency in a focal demyelination model. In summary, while FABP7 is important in OPC differentiation in vitro, its function is not crucial for myelination and remyelination in vivo.

Metformin Restores CNS Remyelination Capacity by Rejuvenating Aged Stem Cells.

The age-related failure to produce oligodendrocytes from oligodendrocyte progenitor cells (OPCs) is associated with irreversible neurodegeneration in multiple sclerosis (MS). Consequently, regenerative approaches have significant potential for treating chronic demyelinating diseases. Here, we show that the differentiation potential of adult rodent OPCs decreases with age. Aged OPCs become unresponsive to pro-differentiation signals, suggesting intrinsic constraints on therapeutic approaches aimed at enhancing OPC differentiation. This decline in functional capacity is associated with hallmarks of cellular aging, including decreased metabolic function and increased DNA damage. Fasting or treatment with metformin can reverse these changes and restore the regenerative capacity of aged OPCs, improving remyelination in aged animals following focal demyelination. Aged OPCs treated with metformin regain responsiveness to pro-differentiation signals, suggesting synergistic effects of rejuvenation and pro-differentiation therapies. These findings provide insight into aging-associated remyelination failure and suggest therapeutic interventions for reversing such declines in chronic disease.

Diversity in the oligodendrocyte lineage: Plasticity or heterogeneity?

Heterogeneity is a widely recognized phenomenon within the majority of cell types in the body including cells of the central nervous system (CNS). The heterogeneity of neurons based on their distinct transmission modes and firing patterns has been recognized for decades, and is necessary to coordinate the immense variety of functions of the CNS. More recently, heterogeneity in glial cells has been identified, including heterogeneity in oligodendrocyte progenitor cells (OPCs) and oligodendrocytes. OPC subpopulations have been described based on their developmental origin, anatomical location in the grey or white matter, and expression of surface receptors. Oligodendrocytes are categorised according to differences in gene expression, myelinogenic potential, and axon specificity. Much of what is described as heterogeneity in oligodendrocyte lineage cells (OLCs) is based on phenotypic differences. However, without evidence for functional differences between putative subgroups of OLCs, distinguishing heterogeneity from plasticity and lineage state is difficult. Identifying functional differences between phenotypically distinct groups are therefore necessary for a deeper understanding of the role of OLCs in health and disease.

Linking NRP2 With EMT and Chemoradioresistance in Bladder Cancer.

Neuropilin-2 (NRP2) is a prognostic indicator for reduced survival in bladder cancer (BCa) patients. Together with its major ligand, vascular endothelial growth factor (VEGF)-C, NRP2 expression is a predictive factor for treatment outcome in response to radiochemotherapy in BCa patients who underwent transurethral resection. Therefore, we investigated the benefit of combining cisplatin-based chemotherapy with irradiation treatment in the BCa cell line RT112 exhibiting or lacking endogenous NRP2 expression in order to evaluate NRP2 as potential therapeutic target. We have identified a high correlation of NRP2 and the glioma-associated oncogene family zinc finger 2 (GLI2) transcripts in the cancer genome atlas (TCGA) cohort of BCa patients and a panel of 15 human BCa cell lines. Furthermore, we used in vitro BCa models to show the transforming growth factor-beta 1 (TGFß1)-dependent regulation of NRP2 and GLI2 expression levels. Since NRP2 was shown to bind TGFß1, associate with TGFß receptors, and enhance TGFß1 signaling, we evaluated downstream signaling pathways using an epithelial-to-mesenchymal transition (EMT)-assay in combination with a PCR profiling array containing 84 genes related to EMT. Subsequent target validation in NRP2 knockout and knockdown models revealed secreted phosphoprotein 1 (SPP1/OPN/Osteopontin) as a downstream target positively regulated by NRP2.

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.

EGFRvIII mutations can emerge as late and heterogenous events in glioblastoma development and promote angiogenesis through Src activation.

BACKGROUND: Amplification of the epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are among the most common genetic alterations in glioblastoma (GBM), the most frequent and most aggressive primary brain tumor. METHODS: In the present work, we analyzed the clonal evolution of these major EGFR aberrations in a small cohort of GBM patients using a unique surgical multisampling technique. Furthermore, we overexpressed both receptors separately and together in 2 patient-derived GBM stem cell lines (GSCs) to analyze their functions in vivo in orthotopic xenograft models. RESULTS: In human GBM biopsies, we identified EGFR amplification as an early event because EGFRvIII mutations emerge from intratumoral heterogeneity later in tumor development. To investigate the biological relevance of this distinct developmental pattern, we established experimental model systems. In these models, EGFR+ tumor cells showed activation of classical downstream signaling pathways upon EGF stimulation and displayed enhanced invasive growth without evidence of angiogenesis in vivo. In contrast, EGFRvIII+ tumors were driven by activation of the prototypical Src family kinase c-Src that promoted VEGF secretion leading to angiogenic tumor growth. CONCLUSIONS: The presented work shows that sequential EGFR amplification and EGFRvIII mutations might represent concerted evolutionary events that drive the aggressive nature of GBM by promoting invasion and angiogenesis via distinct signaling pathways. In particular, c-SRC may be an attractive therapeutic target for tumors harboring EGFRvIII as we identified this protein specifically mediating angiogenic tumor growth downstream of EGFRvIII.

Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer - Proof of concept towards a systems pharmacology approach.

Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006rERLO0.5 adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006rERLO0.5 cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006rERLO0.5 cells. Taken together, differential proteins in the EGFR interactome of HCC4006rERLO0.5 cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance.

Pre-Existing Mature Oligodendrocytes Do Not Contribute to Remyelination following Toxin-Induced Spinal Cord Demyelination.

Remyelination is the regenerative response to demyelination. Although the oligodendrocyte progenitor is established as the major source of remyelinating cells, there is no conclusive evidence on whether mature, differentiated oligodendrocytes can also contribute to remyelination. Using two different inducible myelin-CreER mouse strains in which mature oligodendrocytes were prelabeled by the expression of membrane-bound Green fluorescent protein, we found that after focal spinal cord demyelination, the surrounding surviving labeled oligodendrocytes did not proliferate but remained at a consistent density. Furthermore, existing (prelabeled) oligodendrocytes showed no evidence of incorporation or migration into the lesioned area, or of process extension from the peripheral margins into the lesion. Thus, mature oligodendrocytes do not normally contribute to remyelination and are therefore not a promising target for regenerative therapy.

Integration of Hedgehog and mutant FLT3 signaling in myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations resulting in constitutive kinase activity are common in acute myeloid leukemia (AML) and carry a poor prognosis. Several agents targeting FLT3 have been developed, but their limited clinical activity suggests that the inhibition of other factors contributing to the malignant phenotype is required. We examined gene expression data sets as well as primary specimens and found that the expression of GLI2, a major effector of the Hedgehog (Hh) signaling pathway, was increased in FLT3-ITD compared to wild-type FLT3 AML. To examine the functional role of the Hh pathway, we studied mice in which Flt3-ITD expression results in an indolent myeloproliferative state and found that constitutive Hh signaling accelerated the development of AML by enhancing signal transducer and activator of transcription 5 (STAT5) signaling and the proliferation of bone marrow myeloid progenitors. Furthermore, combined FLT3 and Hh pathway inhibition limited leukemic growth in vitro and in vivo, and this approach may serve as a therapeutic strategy for FLT3-ITD AML.

Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics.

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR-binding proteins are required to elucidate this network. We affinity-purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high-resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc-1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well-described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin-associated adaptor complex AP-2 showed 4.6-fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP-2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.

Endothelial progenitor cells in chronic renal insufficiency.

There is growing evidence for a role of endothelial progenitor cells (EPCs) in the repair of damaged endothelium. It remains unclear which cell populations are most useful for clinical trials. Administration of drugs increasing EPC numbers and/or improving functional properties seems attractive. Further basic research is necessary to understand the mechanisms of mobilization, differentiation and homing of EPC in general and in particular under uremic conditions. Nephrologists should search for strategies to ameliorate EPC dysfunction of uremia. In this way it might be possible to test whether improved EPC biology is associated with decreased cardiovascular mortality in uremic humans. In any such studies the difficulties are going to be related to the complex procedures for EPC isolation, the testing of their identity and differentiation and their propagation before use.

Increased total number but impaired migratory activity and adhesion of endothelial progenitor cells in patients on long-term hemodialysis.

BACKGROUND: Endothelial progenitor cells (EPCs), derived from bone marrow, contribute to vessel repair and neovascularization. Because uremia is a state of endothelial dysfunction associated with high cardiovascular mortality, as well as a state of reduced hematopoiesis, we studied the number and function of EPCs in patients on long-term hemodialysis (HD) therapy. METHODS: We counted the number of EPCs in 20 HD patients and 16 healthy volunteers. To assess EPC function, we measured migratory activity, adhesion to matrix proteins, and adhesion to endothelial cells. Furthermore, we measured blood levels of vascular endothelial growth factor (VEGF) and granulocyte-macrophage colony-stimulating factor, factors known to influence EPC kinetics. Circulating precursor cells (CD34+ , CD34+ /CD133+ , CD34+ /KDR+ cells) were counted by means of flow cytometric analysis. RESULTS: The number of EPCs in HD patients was significantly elevated compared with controls (459.7 +/- 92 versus 364.8 +/- 77.4 EPC/high-power field). However, migratory activity was markedly decreased in HD patients (47.5 +/- 27.7 versus 84.7 +/- 3.2 EPC/high-power field). EPCs of HD patients showed impaired adhesion to extracellular matrix and endothelial cells. VEGF blood levels in HD patients were 2-fold greater compared with controls. The number of circulating CD34+ and CD34+ /133+ cells was reduced in HD patients. There were no differences in total numbers of CD34+ /KDR+ cells. CONCLUSION: This study shows an elevated number, but pronounced functional impairment, of EPCs in patients on long-term HD therapy. The latter may result in limited endothelial repair, which, in turn, may contribute to endothelial dysfunction in this particular group of patients.