Frontline Science: The expression of integrin αD ß2 (CD11d/CD18) on neutrophils orchestrates the defense mechanism against endotoxemia and sepsis.

Neutrophil-macrophage interplay is a fine-tuning mechanism that regulates the innate immune response during infection and inflammation. Cell surface receptors play an essential role in neutrophil and macrophage functions. The same receptor can provide different outcomes within diverse leukocyte subsets in different inflammatory conditions. Understanding the variety of responses mediated by one receptor is critical for the development of anti-inflammatory treatments. In this study, we evaluated the role of a leukocyte adhesive receptor, integrin αD ß2 , in the development of acute inflammation. αD ß2 is mostly expressed on macrophages and contributes to the development of chronic inflammation. In contrast, we found that αD -knockout dramatically increases mortality in the cecal ligation and puncture sepsis model and LPS-induced endotoxemia. This pathologic outcome of αD -deficient mice is associated with a reduced number of monocyte-derived macrophages and an increased number of neutrophils in their lungs. However, the tracking of adoptively transferred fluorescently labeled wild-type (WT) and αD-/- monocytes in WT mice during endotoxemia demonstrated only a moderate difference between the recruitment of these two subsets. Moreover, the rescue experiment, using i.v. injection of WT monocytes to αD -deficient mice followed by LPS challenge, showed only slightly reduced mortality. Surprisingly, the injection of WT neutrophils to the bloodstream of αD-/- mice markedly increased migration of monocyte-derived macrophage to lungs and dramatically improves survival. αD -deficient neutrophils demonstrate increased necrosis/pyroptosis. αD ß2 -mediated macrophage accumulation in the lungs promotes efferocytosis that reduced mortality. Hence, integrin αD ß2 implements a complex defense mechanism during endotoxemia, which is mediated by macrophages via a neutrophil-dependent pathway.

Inhibition of focal adhesion kinase increases adult olfactory stem cell self-renewal and neuroregeneration through ciliary neurotrophic factor.

Constant neuroregeneration in adult olfactory epithelium maintains olfactory function by basal stem cell proliferation and differentiation to replace lost olfactory sensory neurons (OSNs). Understanding the mechanisms regulating this process could reveal potential therapeutic targets for stimulating adult olfactory neurogenesis under pathological conditions and aging. Ciliary neurotrophic factor (CNTF) in astrocytes promotes forebrain neurogenesis but its function in the olfactory system is unknown. Here, we show in mouse olfactory epithelium that CNTF is expressed in horizontal basal cells, olfactory ensheathing cells (OECs) and a small subpopulation of OSNs. CNTF receptor alpha was expressed in Mash1-positive globose basal cells (GBCs) and OECs. Thus, CNTF may affect GBCs in a paracrine manner. CNTF-/- mice did not display altered GBC proliferation or olfactory function, suggesting that CNTF is not involved in basal olfactory renewal or that they developed compensatory mechanisms. Therefore, we tested the effect of increased CNTF in wild type mice. Intranasal instillation of a focal adhesion kinase (FAK) inhibitor, FAK14, upregulated CNTF expression. FAK14 also promoted GBC proliferation, neuronal differentiation and basal stem cell self-renewal but had no effective in CNTF-/- mice, suggesting that FAK inhibition promotes olfactory neuroregeneration through CNTF, making them potential targets to treat sensorineural anosmia due to OSN loss.

P/Q Type Calcium Channel Cav2.1 Defines a Unique Subset of Glomeruli in the Mouse Olfactory Bulb.

Voltage-gated calcium (Cav) channels are a prerequisite for signal transmission at the first olfactory sensory neuron (OSN) synapse within the glomeruli of the main olfactory bulb (MOB). We showed previously that the N-type Cav channel subunit Cav2.2 is present in the vast majority of glomeruli and plays a central role in presynaptic transmitter release. Here, we identify a distinct subset of glomeruli in the MOB of adult mice that is characterized by expression of the P/Q-type channel subunit Cav2.1. Immunolocalization shows that Cav2.1+ glomeruli reside predominantly in the medial and dorsal MOB, and in the vicinity of the necklace glomerular region close to the accessory olfactory bulb. Few glomeruli are detected on the ventral and lateral MOB. Cav2.1 labeling in glomeruli colocalizes with the presynaptic marker vGlut2 in the axon terminals of OSNs. Electron microscopy shows that Cav2.1+ presynaptic boutons establish characteristic asymmetrical synapses with the dendrites of second-order neurons in the glomerular neuropil. Cav2.1+ glomeruli receive axonal input from OSNs that express molecules of canonical OSNs: olfactory marker protein, the ion channel Cnga2, and the phosphodiesterase Pde4a. In the main olfactory epithelium, Cav2.1 labels a distinct subpopulation of OSNs whose distribution mirrors the topography of the MOB glomeruli, that shows the same molecular signature, and is already present at birth. Together, these experiments identify a unique Cav2.1+ multiglomerular domain in the MOB that may form a previously unrecognized olfactory subsystem distinct from other groups of necklace glomeruli that rely on cGMP signaling mechanisms.

Plasma membrane insertion of KCa2.3 (SK3) is dependent upon the SNARE proteins, syntaxin-4 and SNAP23.

We previously demonstrated endocytosis of KCa2.3 is caveolin-1-, dynamin II- and Rab5-dependent. KCa2.3 then enters Rab35/EPI64C- and RME-1-containing recycling endosomes and is returned to the plasma membrane (PM). Herein, we report on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate KCa2.3 colocalizes with SNAP-23 and Syntaxin-4 in the PM of HEK and endothelial cells by confocal immunofluorescence microscopy. We further show KCa2.3 can be co-immunoprecipitated with SNAP-23 and Syntaxin-4. Overexpression of either Syntaxin-4 or SNAP-23 increased PM expression of KCa2.3, whereas shRNA-mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression, as assessed by cell surface biotinylation. Whole-cell patch clamp studies confirmed knockdown of SNAP-23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate shRNA mediated knockdown of SNAP-23 inhibits recycling of KCa2.3 following endocytosis, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide (BLAP)-tagged KCa2.3, coupled with ER-resident biotin ligase (BirA), channels could be biotinylated in the ER after which we evaluated their rate of insertion into the PM following Golgi exit. We demonstrate knockdown of SNAP-23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized KCa2.3 channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin-4 and SNAP-23.

Development of a Function-Blocking Antibody Against Fibulin-3 as a Targeted Reagent for Glioblastoma.

Purpose: We sought a novel approach against glioblastomas (GBM) focused on targeting signaling molecules localized in the tumor extracellular matrix (ECM). We investigated fibulin-3, a glycoprotein that forms the ECM scaffold of GBMs and promotes tumor progression by driving Notch and NFκB signaling.Experimental Design: We used deletion constructs to identify a key signaling motif of fibulin-3. An mAb (mAb428.2) was generated against this epitope and extensively validated for specific detection of human fibulin-3. mAb428.2 was tested in cultures to measure its inhibitory effect on fibulin-3 signaling. Nude mice carrying subcutaneous and intracranial GBM xenografts were treated with the maximum achievable dose of mAb428.2 to measure target engagement and antitumor efficacy.Results: We identified a critical 23-amino acid sequence of fibulin-3 that activates its signaling mechanisms. mAb428.2 binds to that epitope with nanomolar affinity and blocks the ability of fibulin-3 to activate ADAM17, Notch, and NFκB signaling in GBM cells. mAb428.2 treatment of subcutaneous GBM xenografts inhibited fibulin-3, increased tumor cell apoptosis, and enhanced the infiltration of inflammatory macrophages. The antibody reduced tumor growth and extended survival of mice carrying GBMs as well as other fibulin-3-expressing tumors. Locally infused mAb428.2 showed efficacy against intracranial GBMs, increasing tumor apoptosis and reducing tumor invasion and vascularization, which are enhanced by fibulin-3.Conclusions: To our knowledge, this is the first rationally developed, function-blocking antibody against an ECM target in GBM. Our results offer a proof of principle for using "anti-ECM" strategies toward more efficient targeted therapies for malignant glioma. Clin Cancer Res; 24(4); 821-33. ©2017 AACR.

Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons.

Odorant receptors (OR) are strongly implicated in coalescence of olfactory sensory neuron (OSN) axons and the formation of olfactory bulb (OB) glomeruli. However, when ORs are first expressed relative to basal cell division and OSN axon extension is unknown. We developed an in vivo fate-mapping strategy that enabled us to follow OSN maturation and axon extension beginning at basal cell division. In parallel, we mapped the molecular development of OSNs beginning at basal cell division, including the onset of OR expression. Our data show that ORs are first expressed around 4 d following basal cell division, 24 h after OSN axons have reached the OB. Over the next 6+ days the OSN axons navigate the OB nerve layer and ultimately coalesce in glomeruli. These data provide a previously unidentified perspective on the role of ORs in homophilic OSN axon adhesion and lead us to propose a new model dividing axon extension into two phases. Phase I is OR-independent and accounts for up to 50% of the time during which axons approach the OB and begin navigating the olfactory nerve layer. Phase II is OR-dependent and concludes as OSN axons coalesce in glomeruli.

Novel paracrine modulation of Notch-DLL4 signaling by fibulin-3 promotes angiogenesis in high-grade gliomas.

High-grade gliomas are characterized by exuberant vascularization, diffuse invasion, and significant chemoresistance, resulting in a recurrent phenotype that makes them impossible to eradicate in the long term. Targeting protumoral signals in the glioma microenvironment could have significant impact against tumor cells and the supporting niche that facilitates their growth. Fibulin-3 is a protein secreted by glioma cells, but absent in normal brain, that promotes tumor invasion and survival. We show here that fibulin-3 is a paracrine activator of Notch signaling in endothelial cells and promotes glioma angiogenesis. Fibulin-3 overexpression increased tumor VEGF levels, microvascular density, and vessel permeability, whereas fibulin-3 knockdown reduced vessel density in xenograft models of glioma. Fibulin-3 localization in human glioblastomas showed dense fiber-like condensations around tumor blood vessels, which were absent in normal brain, suggesting a remarkable association of this protein with tumor endothelium. At the cellular level, fibulin-3 enhanced endothelial cell motility and association to glioma cells, reduced endothelial cell sprouting, and increased formation of endothelial tubules in a VEGF-independent and Notch-dependent manner. Fibulin-3 increased ADAM10/17 activity in endothelial cells by inhibiting the metalloprotease inhibitor TIMP3; this resulted in increased Notch cleavage and increased expression of DLL4 independently of VEGF signaling. Inhibition of ADAM10/17 or knockdown of DLL4 reduced the proangiogenic effects of fibulin-3 in culture. Taken together, these results reveal a novel, proangiogenic role of fibulin-3 in gliomas, highlighting the relevance of this protein as an important molecular target in the tumor microenvironment.

Aging in the olfactory system.

With advancing age, the ability of humans to detect and discriminate odors declines. In light of the rapid progress in analyzing molecular and structural correlates of developing and adult olfactory systems, the paucity of information available on the aged olfactory system is startling. A rich literature documents the decline of olfactory acuity in aged humans, but the underlying cellular and molecular mechanisms are largely unknown. Using animal models, preliminary work is beginning to uncover differences between young and aged rodents that may help address the deficits seen in humans, but many questions remain unanswered. Recent studies of odorant receptor (OR) expression, synaptic organization, adult neurogenesis, and the contribution of cortical representation during aging suggest possible underlying mechanisms and new research directions.

Dishevelled proteins are associated with olfactory sensory neuron presynaptic terminals.

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.

Renal cystic disease proteins play critical roles in the organization of the olfactory epithelium.

It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases - polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) - localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (G(olf), AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.

Chromosomal location-dependent nonstochastic onset of odor receptor expression.

As odorant receptors (ORs) are thought to be critical determinants of olfactory sensory neuron (OSN) axon targeting and organization, we examined the spatiotemporal onset of mice ORs expression from the differentiation of OSNs in the olfactory placode to an aging olfactory epithelium. ORs were first detected in the placode at embryonic day 9 (E9), at the onset of OSN differentiation but before axon extension. By E13, 22 of 23 ORs were expressed. Onset of individual OR expression was diverse; levels and patterns of expression were unique for each OR. Regional distribution of ORs within zones of the olfactory epithelium appeared stable across development; adult-like patterns were observed by E13. Finally, analysis of OR expression and chromosomal location suggests that ORs are not stochastically expressed; they show evidence of coordinated expression. Collectively, these studies demonstrate that ORs are not equally represented in the "olfactome" across an animal's lifespan.

Onset of odorant receptors.

Odorant receptors are thought to be critical determinants of olfactory sensory neuron axon targeting and organization. Nonetheless, a systematic characterization of the onset of odorant receptor expression has not yet been done in the main olfactory epithelium. Here, we briefly review our current understanding regarding the onset of odorant receptor expression in the main olfactory epithelium and identify some of those questions which we believe must be of high priority for future study.

Blood vessels form a migratory scaffold in the rostral migratory stream.

In adult mice, new neurons born in the subventricular zone (SVZ), lining the lateral ventricles, migrate tangentially into the olfactory bulb along a well-delineated path, the rostral migratory stream (RMS). Neuroblasts in the RMS migrate tangentially in chains, without a recognized migratory scaffold. Here we quantitatively examine the distribution of, and relationships between, cells within the RMS, throughout its rostral-caudal extent. We show that there is a higher density of blood vessels in the RMS than in other brain regions, including areas with equal cell density, and that the orientation of blood vessels parallels the RMS throughout the caudal to rostral path. Of particular interest, migratory neuroblast chains are longitudinally aligned along blood vessels within the RMS, with over 80% of vessel length in rostral areas of the RMS apposed by neuroblasts. Electron micrographs show direct contact between endothelial cells and neuroblasts, although intervening astrocytic processes are often present. Within the RMS, astrocytes arborize extensively, extending long processes that are parallel to blood vessels and the direction of neuroblast migration. Thus, the astrocytic processes establish a longitudinal alignment within the RMS, rather than a more typical stellate shape. This complementary alignment suggests that blood vessels and astrocytes may cooperatively establish a scaffold for migrating neuroblasts, as well as provide and regulate migratory cues.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Functional expression of the olfactory signaling system in the kidney.

Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.

Wnt/Frizzled family members mediate olfactory sensory neuron axon extension.

A comprehensive model has yet to emerge, but it seems likely that numerous mechanisms contribute to the specificity of olfactory sensory neuron (OSN) axon innervation of the olfactory bulb. Elsewhere in the nervous system the Wnt/Fz family has been implicated in patterning of anterior-posterior axes, cell type specification, cell proliferation, and axon guidance. Because of our work describing cadherin-catenin family member expression in the primary olfactory pathway, and because mechanisms of Wnt-Fz interactions can depend in part on catenins, we were encouraged to explore Wnt-Fz expression and function in OSN axon extension. Here, we show that OSNs express Fz-1, Fz-3, and Wnt-5a, whereas olfactory ensheathing cells (OECs) express Wnt-4. Fz-7 is also expressed in the olfactory nerve by cells that delineate large axon fascicles, but are negative for OEC markers. Fz-1 showed a developmental downregulation. However, in adults it is expressed at different levels across the olfactory epithelium and in restricted glomeruli across the olfactory bulb, suggesting an important role in the formation and maintenance of OSN connections to the olfactory bulb. Reporter TOPGAL mice demonstrated that some OECs located in the inner olfactory nerve layer can respond to Wnt ligands. Of further interest, we show here with in vitro assays that Wnt-5a increases OSN axon outgrowth and alters growth cone morphology. Our data point to a key role for Wnt/Fz molecules in the development of the mouse olfactory system, providing complementary mechanisms required for OSN axon extension and coalescence.

Acute hypoxia differentially affects the gamma-aminobutyric acid type A receptor alpha(1), alpha(2), beta(2), and gamma(2) subunit mRNA levels in the developing chick optic tectum: stage-dependent sensitivity.

This investigation analyzes the effect of an acute hypoxic treatment on the level of four (alpha(1), alpha(2), beta(2), and gamma(2)) subunit mRNAs of the GABA(A) receptor in layer "i" of the developing chick optic tectum. Our results show that 1 hr of normobaric acute hypoxia significantly changes the subunit mRNA levels. Different subunit mRNAs display different sensitivity to hypoxia: alpha(1), beta(2), and gamma(2) mRNAs are highly sensitive, whereas alpha(2) mRNA is almost not affected. The sensitivity of the mRNA levels to hypoxia is stage dependent. The mean percentages of variation produced by the hypoxia in the level of expression of the four subunits were 20% at ED12, 5% at ED16, and only 2% at ED18. These changes in the mean percentages of expression modify the probability of coexpression. In the case of double mRNA combinations, the hypoxia produced a mean variation in the probability of coexpression of 37% at ED12, 8% at ED16, and only 4% at ED18. With regard to the triple subunit mRNAs combinations, the variations were 206% at ED12, 11% at ED16, and only 7% at ED18. The quadruple combination values were 1,500% at ED12, 21% at ED16, and only 11% at ED18. This study demonstrates that the subunit mRNA levels are highly sensitive during the early stages, suggesting that GABA(A) receptor composition might undergo environment-dependent plastic changes providing a high degree of plasticity to the GABA neurotransmitter system development.

Development and localisation of GABA(A) receptor alpha1, alpha2, beta2 and gamma2 subunit mRNA in the chick optic tectum.

An in situ hybridisation technique was used to analyse the spatial and temporal pattern of expression of the mRNA encoding the four gamma-aminobutyric acid A (GABA(A)) receptor subunits (alpha1, alpha2, beta2, and gamma2) in the developing chick optic tectum. As a rule, layer i, layer h, and transient cell compartment 3 (TCC3) show the highest levels of expression, especially of alpha1, alpha2 and beta2, which undergo striking changes as a function of time. Apart from these common features, the global pattern is highly complex and dynamic. Such complexity derives from the fact that each subunit exhibits a characteristically distinct pattern of expression and the temporal evolution of each differs in the different layers of the tectum. The influence of several developmental cell behaviours such as proliferation, neuronal migration, programmed cell death, and differentiation must be taken into account to understand pattern complexity and dynamics. Our results suggest that differences in the rate of subunit expression, particularly of alpha1, alpha2, and beta2, could have significant consequences on GABA(A) receptor complex subunit composition along development and on the functional properties of the GABA neurotransmitter system.