Summary Two cDNAs encoding allene oxide cyclases (PpAOC1, PpAOC2), key enzymes in the formation of jasmonic acid (JA) and its precursor (9S,13S)‐12‐oxo‐phytodienoic acid (cis‐(+)‐OPDA), were isolated from the moss Physcomitrella patens. Recombinant PpAOC1 and PpAOC2 show substrate specificity against the allene oxide derived from 13‐hydroperoxy linolenic acid (13‐HPOTE); PpAOC2 also shows substrate specificity against the allene oxide derived from 12‐hydroperoxy arachidonic acid (12‐HPETE). In protonema and gametophores the occurrence of cis‐(+)‐OPDA, but neither JA nor the isoleucine conjugate of JA nor that of cis‐(+)‐OPDA was detected. Targeted knockout mutants for PpAOC1 and for PpAOC2 were generated, while double mutants could not be obtained. The ΔPpAOC1 and ΔPpAOC2 mutants showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis.
Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.MRI | nanoparticle imaging | USPIO | multiple sclerosis | EAE
It is known that knockdown of the mitochondrial 18 kDa translocator protein (TSPO) as well as TSPO ligands modulate various functions, including functions related to cancer. To study the ability of TSPO to regulate gene expression regarding such functions, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 15 min, the classical TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. These changes also included gene products that are part of the canonical pathway serving to modulate general gene expression. These changes are in accord with real-time, reverse transcriptase (RT) PCR. At the time points of 15, 30, 45, and 60 min, as well as 3 and 24 h of PK 11195 exposure, the functions associated with the changes in gene expression in these glioblastoma cells covered well known TSPO functions. These functions included cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was corroborated microscopically for cell migration, cell accumulation, adhesion, and neuronal differentiation. Changes in gene expression at 24 h of PK 11195 exposure were related to downregulation of tumorigenesis and upregulation of programmed cell death. In the vehicle treated as well as PK 11195 exposed cell cultures, our triple labeling showed intense TSPO labeling in the mitochondria but no TSPO signal in the cell nuclei. Thus, mitochondrial TSPO appears to be part of the mitochondria-to-nucleus signaling pathway for modulation of nuclear gene expression. The novel TSPO ligand 2-Cl-MGV-1 appeared to be very specific regarding modulation of gene expression of immediate early genes and transcription factors.
Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.
By exposing cells of the U118MG glioblastoma cell line to protoporphyrin IX (PPIX) in culture, we found that the 18 kDa mitochondrial translocator protein (TSPO) prevents intracellular accumulation of PPIX. In particular, TSPO knockdown by stable transfection of TSPO silencing siRNA vectors into U118MG cells leads to mitochondrial PPIX accumulation. In combination with light exposure, the PPIX accumulation led to cell death of the TSPO knockdown cells. In the sham control cells (stable transfection of scrambled siRNA vectors), TSPO expression remained high and no PPIX accumulation was observed. The prevention of PPIX accumulation by TSPO was not due to conversion of PPIX to heme in the sham control cells. Similar to TSPO knockdown, the reactive oxygen species (ROS) scavenger glutathione (GSH) also enhanced PPIX accumulation. This suggests that that ROS generation as modulated by TSPO activation may present a mechanism to prevent accumulation of PPIX.
Intact TSPO appears to be able to counteract the invasive and angiogenic characteristics related to the aggressiveness of U118MG glioblastoma cells in vivo and in vitro.
Glioblastoma multiforme alters healthy tissue vasculature by inducing angiogenesis and vascular remodeling. To fully comprehend the structural and functional properties of the resulting vascular network, it needs to be studied collectively by considering both geometric and topological properties. Utilizing Single Plane Illumination Microscopy (SPIM), the detailed capillary structure in entire healthy and tumor-bearing mouse brains could be resolved in three dimensions. At the scale of the smallest capillaries, the entire vascular systems of bulk U87- and GL261-glioblastoma xenografts, their respective cores, and healthy brain hemispheres were modeled as complex networks and quantified with fundamental topological measures. All individual vessel segments were further quantified geometrically and modular clusters were uncovered and characterized as meta-networks, facilitating an analysis of large-scale connectivity. An inclusive comparison of large tissue sections revealed that geometric properties of individual vessels were altered in glioblastoma in a relatively subtle way, with high intra- and inter-tumor heterogeneity, compared to the impact on the vessel connectivity. A network topology analysis revealed a clear decomposition of large modular structures and hierarchical network organization, while preserving most fundamental topological classifications, in both tumor models with distinct growth patterns. These results augment our understanding of cerebrovascular networks and offer a topological assessment of glioma-induced vascular remodeling. The findings may help understand the emergence of hypoxia and necrosis, and prove valuable for therapeutic interventions such as radiation or antiangiogenic therapy.
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