Abstract Peripheral or central nerve injury is a frequent cause of chronic pain and the mechanisms are not fully understood. Using newly generated transgenic mice we show that progranulin ...overexpression in sensory neurons attenuates neuropathic pain after sciatic nerve injury and accelerates nerve healing. A yeast-2-hybrid screen revealed putative interactions of progranulin with autophagy-related proteins, ATG12 and ATG4b. This was supported by colocalization and proteomic studies showing regulations of ATG13 and ATG4b and other members of the autophagy network, lysosomal proteins and proteins involved in endocytosis. The association of progranulin with the autophagic pathway was functionally confirmed in primary sensory neurons. Autophagy and survival were impaired in progranulin-deficient neurons and improved in progranulin overexpressing neurons. Nerve injury in vivo caused an accumulation of LC3b-EGFP positive bodies in neurons of the dorsal root ganglia and nerves suggesting an impairment of autophagic flux. Overexpression of progranulin in these neurons was associated with a reduction of the stress marker ATF3, fewer protein aggregates in the injured nerve and enhanced stump healing. At the behavioral level, further inhibition of the autophagic flux by hydroxychloroquine intensified cold and heat nociception after sciatic nerve injury and offset the pain protection provided by progranulin. We infer that progranulin may assist in removal of protein waste and thereby helps to resolve neuropathic pain after nerve injury.
Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the ...presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP
tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior.
Blood vessels form de novo through the tightly regulated programs of vasculogenesis and angiogenesis. Both processes are distinct but one of the steps they share is the formation of a central lumen, ...when groups of cells organized as vascular cords undergo complex changes to achieve a tube-like morphology. Recently, a protein termed epidermal growth factor-like domain 7 (EGFL7) was described as a novel endothelial cell-derived factor involved in the regulation of the spatial arrangement of cells during vascular tube assembly. With its impact on tubulogenesis and vessel shape EGFL7 joined the large family of molecules governing blood vessel formation. Only recently, the molecular mechanisms underlying EGFL7's effects have been started to be elucidated and shaping of the extracellular matrix (ECM) as well as Notch signaling might very well play a role in mediating its biological effects. Further, findings in knock-out animal models suggest miR-126, a miRNA located within the egfl7 gene, has a major role in vessel development by promoting VEGF signaling, angiogenesis and vascular integrity. This review summarizes our current knowledge on EGFL7 and miR-126 and we will discuss the implications of both bioactive molecules for the formation of blood vessels.
OBJECTIVE—Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. ...Here we studied the role of DMBT1 in endothelial cells.
METHODS AND RESULTS—DMBT1 was secreted into the extracellular matrix (ECM) by endothelial cells in vitro and in situ and the presence of DMBT1 in the ECM increased endothelial cell adherence. Endothelial cell-derived DMBT1 associated with galectin-3 (coprecipitation), and human recombinant DMBT1 bound EGF, vascular endothelial growth factor and Delta-like (Dll) 4 (specific ELISAs). Compared to cells from wild-type mice, endothelial cells from DMBT1 mice demonstrated reduced migration, proliferation, and tube formation. In vivo recovery from hindlimb ischemia was attenuated in DMBT1 animals as was vascular endothelial growth factor -induced endothelial sprouting from isolated aortic rings; the latter response could be rescued by the addition of recombinant DMBT1. The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation and we found that endothelial cells from DMBT1 mice expressed more EphrinB2 than cells from wild-type mice. Levels of Dll1, Dll4, Hes1, Hey1, and EphB4, on the other hand, were decreased.
CONCLUSION—Taken together, the results of this study indicate that DMBT1 functions as an important endothelium-derived ECM protein that is able to bind angiogenic factors and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4.
Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have ...shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of γ radiation. GPCs were treated with 2.5 Gy of γ radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape.
A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We ...found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.
Adult neural stem cells reside in a specialized niche in the subventricular zone (SVZ). Throughout life they give rise to adult-born neurons in the olfactory bulb (OB), thus contributing to neural ...plasticity and pattern discrimination. Here, we show that the neurovascular protein EGFL7 is secreted by endothelial cells and neural stem cells (NSCs) of the SVZ to shape the vascular stem-cell niche. Loss of EGFL7 causes an accumulation of activated NSCs, which display enhanced activity and re-entry into the cell cycle. EGFL7 pushes activated NSCs towards quiescence and neuronal progeny towards differentiation. This is achieved by promoting Dll4-induced Notch signalling at the blood vessel-stem cell interface. Fewer inhibitory neurons form in the OB of EGFL7-knockout mice, which increases the absolute signal conducted from the mitral cell layer of the OB but decreases neuronal network synchronicity. Consequently, EGFL7-knockout mice display severe physiological defects in olfactory behaviour and perception.
Communication signals and signaling pathways are often studied in different physiological systems. However, it has become abundantly clear that the immune system is not self-regulated, but functions ...in close association with the nervous system. The neural–immune interface is complex; its balance determines cancer progression, as well as autoimmune disorders. Immunotherapy remains a promising approach in the context of glioblastoma multiforme (GBM). The primary obstacle to finding effective therapies is the potent immunosuppression induced by GBM. Anti-inflammatory cytokines, induction of regulatory T cells, and the expression of immune checkpoint molecules are the key mediators for immunosuppression in the tumor microenvironment. Immune checkpoint molecules are ligand–receptor pairs that exert inhibitory or stimulatory effects on immune responses. In the past decade, they have been extensively studied in preclinical and clinical trials in diseases such as cancer or autoimmune diseases in which the immune system has failed to maintain homeostasis. In this review, we will discuss promising immune-modulatory targets that are in the focus of current clinical research in glioblastoma, but are also in the precarious position of potentially becoming starting points for the development of autoimmune diseases like multiple sclerosis.
The localization, expression, and physiological role of regulatory proteins in the neurogenic niches of the brain is fundamental to our understanding of adult neurogenesis. This study explores the ...expression and role of the E3-ubiquitin ligase, c-Cbl, in neurogenesis within the subventricular zone (SVZ) of mice. In vitro neurosphere assays and in vivo analyses were performed in specific c-Cbl knock-out lines to unravel c-Cbl’s role in receptor tyrosine kinase signaling, including the epidermal growth factor receptor (EGFR) pathway. Our findings suggest that c-Cbl is significantly expressed within EGFR-expressing cells, playing a pivotal role in neural stem cell proliferation and differentiation. However, c-Cbl’s function extends beyond EGFR signaling, as its loss upon knock-out stimulated progenitor cell proliferation in neurosphere cultures. Yet, this effect was not detected in hippocampal progenitor cells, reflecting the lack of the EGFR in the hippocampus. In vivo, c-Cbl exerted only a minor proneurogenic influence with no measurable impact on the formation of adult-born neurons. In conclusion, c-Cbl regulates neural stem cells in the subventricular zone via the EGFR pathway but, likely, its loss is compensated by other signaling modules in vivo.
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