E-cadherin enhances neuregulin signaling and promotes Schwann cell myelination.

In myelinating Schwann cells, E-cadherin is a component of the adherens junctions that stabilize the architecture of the noncompact myelin region. In other cell types, E-cadherin has been considered as a signaling receptor that modulates intracellular signal transduction and cellular responses. To determine whether E-cadherin plays a regulatory role during Schwann cell myelination, we investigated the effects of E-cadherin deletion and over-expression in Schwann cells. In vivo, Schwann cell-specific E-cadherin ablation results in an early myelination delay. In Schwann cell-dorsal root ganglia neuron co-cultures, E-cadherin deletion attenuates myelin formation and shortens the myelin segment length. When over-expressed in Schwann cells, E-cadherin improves myelination on Nrg1 type III(+/-) neurons and induces myelination on normally non-myelinated axons of sympathetic neurons. The pro-myelinating effect of E-cadherin is associated with an enhanced Nrg1-erbB receptor signaling, including activation of the downstream Akt and Rac. Accordingly, in the absence of E-cadherin, Nrg1-signaling is diminished in Schwann cells. Our data also show that E-cadherin expression in Schwann cell is induced by axonal Nrg1 type III, indicating a reciprocal interaction between E-cadherin and the Nrg1 signaling. Altogether, our data suggest a regulatory function of E-cadherin that modulates Nrg1 signaling and promotes Schwann cell myelin formation.

Mammal responses to global changes in human activity vary by trophic group and landscape.

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human-wildlife interactions along gradients of human influence.

Anti-Inflammatory Effects of Encapsulated Human Mesenchymal Stromal/Stem Cells and a Method to Scale-Up Cell Encapsulation.

Mesenchymal stem/stromal cells (MSC) promote recovery in a wide range of animal models of injury and disease. They can act in vivo by differentiating and integrating into tissues, secreting factors that promote cell growth and control inflammation, and interacting directly with host effector cells. We focus here on MSC secreted factors by encapsulating the cells in alginate microspheres, which restrict cells from migrating out while allowing diffusion of factors including cytokines across the capsules. One week after intrathecal lumbar injection of human bone marrow MSC encapsulated in alginate (eMSC), rat IL-10 expression was upregulated in distant rat spinal cord injury sites. Detection of human IL-10 protein in rostrally derived cerebrospinal fluid (CSF) indicated distribution of this human MSC-secreted cytokine throughout rat spinal cord CSF. Intraperitoneal (IP) injection of eMSC in a rat model for endotoxemia reduced serum levels of inflammatory cytokines within 5 h. Detection of human IL-6 in sera after injection of human eMSC indicates rapid systemic distribution of this human MSC-secreted cytokine. Despite proof of concept for eMSC in various disorders using animal models, translation of encapsulation technology has not been feasible primarily because methods for scale-up are not available. To scale-up production of eMSC, we developed a rapid, semi-continuous, capsule collection system coupled to an electrosprayer. This system can produce doses of encapsulated cells sufficient for use in clinical translation.

Public perceptions and attitudes toward urban wildlife encounters - A decade of change.

Europe is currently undergoing dynamic land use changes causing the expansion of urban habitat, which is driving wildlife species to colonise conurbations, resulting in an increased likelihood of human-wildlife conflict (HWC). Understanding people's attitudes toward wildlife is essential to manage these conflicts. This study assessed people's attitudes toward urban wildlife, the types of conflicts that existed, preferences for managing conflict situations, and determined any changes in perceptions of urban wildlife over a decade. A questionnaire survey of residents of Krakow, the second-largest city in Poland, was conducted in 2010 (n = 721) and repeated in 2020 (n = 887). We found that encounters with certain urban wildlife such as wild boars, red squirrels, roe deer, brown hares, and red foxes had increased significantly in 2020 compared to 2010. Respondents reported that wild boar and beavers did not show fear when encountering humans. Stone martens were considered the most nuisance wildlife species in 2010, while in 2020 wild boar were the most conflictual wildlife species. There were additional reports of conflicts with roe deer and red foxes. The most frequent HWC responses were personal anxiety, intrusion into property and destruction of crops, which increased significantly over the decade, independent of respondents' gender. Respondents preferred nonlethal methods to mitigate conflicts. The study provides valuable information and knowledge on changes in people's attitudes toward urban wildlife that can help with wildlife management in urban areas. Incorporating perception and attitude data from the public, along with a multi-stakeholder approach that includes wildlife professionals, in the planning and design of future urban environments is critical to minimise HWC.

Animal-vehicle collisions during the COVID-19 lockdown in early 2020 in the Krakow metropolitan region, Poland.

The interrelations between human activity and animal populations are of increasing interest due to the emergence of the novel COVID-19 and the consequent pandemic across the world. Anthropogenic impacts of the pandemic on animals in urban-suburban environments are largely unknown. In this study, the temporal and spatial patterns of urban animal response to the COVID-19 lockdown were assessed using animal-vehicle collisions (AVC) data. We collected AVC data over two 6-month periods in 2019 and 2020 (January to June) from the largest metropolis in southern Poland, which included lockdown months. Furthermore, we used traffic data to understand the impact of lockdown on AVC in the urban area. Our analysis of 1063 AVC incidents revealed that COVID-19 related lockdown decreased AVC rates in suburban areas. However, in the urban area, even though traffic volume had significantly reduced, AVC did not decrease significantly, suggesting that lockdown did not influence the collision rates in the urban area. Our results suggest that there is a need to focus on understanding the effects of changes in traffic volume on both human behaviour and wildlife space use on the resulting impacts on AVC in the urban area.

Human-Wildlife Conflicts in Krakow City, Southern Poland.

Efforts to reduce human-wildlife-conflict are integral to wildlife management and conservation in urban habitats. In our study, we identified the HWC situations in urban areas of Krakow city, based on animal-vehicle collisions, intrusion to property, and damages. Hot spot analysis and Moran's Index were used to identify the location of maximum potential conflict. We analysed 2512 incidents in which animals (of which 85% included mammals and 15% birds) were involved in conflict situations between 2007 and 2013. A significant seasonal variation was observed among the animals. We also identified roe deer (50.23%), red fox (22.80%) and wild boar (11.40%), as the three prominent conflicted animals. Getis-Ord Gi* analysis was used to identify spatial clusters of conflict. A significant spatial association was found in the location of clusters of hot spots in specific land-use based on Moran's Index. Hot spots of roe deer and wild boar were high in grasslands and in forest and for red fox in built-up area. The results underscore the notion that conservation and wildlife management efforts must take into account differences in the seasonality of HWC among species. This information can be used to inform mitigation strategies.

Differential expression and functions of neuronal and glial neurofascin isoforms and splice variants during PNS development.

The cell adhesion molecule neurofascin (NF) has a major neuronal isoform (NF186) containing a mucin-like domain followed by a fifth fibronectin type III repeat while these domains are absent from glial NF155. Neuronal NF isoforms lacking one or both of these domains are expressed transiently in embryonic dorsal root ganglia (DRG). These two domains are co-expressed in mature NF186, which peaks in expression prior to birth and then persists almost exclusively at nodes of Ranvier on myelinated axons. In contrast, glial NF155 is only detected postnatally with the onset of myelination. All these forms of NF bound homophilically and to Schwann cells but only the mature NF186 isoform inhibits cell adhesion, and this activity may be important in formation of the node of Ranvier. Schwann cells deficient in NF155 myelinated DRG axons in a delayed manner and they showed significantly decreased clustering of both NF and Caspr in regions where paranodes normally form. The combined results suggest that NF186 is expressed prenatally on DRG neurons and it may modulate their adhesive interactions with Schwann cells, which express NF155 postnatally and require it for development of axon-glial paranodal junctions.

E-cadherin expression in postnatal Schwann cells is regulated by the cAMP-dependent protein kinase a pathway.

Expression of E-cadherin in the peripheral nervous system is a highly regulated process that appears postnatally in concert with the development of myelinating Schwann cell lineage. As a major component of autotypic junctions, E-cadherin plays an important role in maintaining the structural integrity of noncompact myelin regions. In vivo, the appearance of E-cadherin in postnatal Schwann cell is accompanied by the disappearance of N-cadherin, suggesting reciprocal regulation of the two cadherins during Schwann cell development. The molecular signal that regulates the cadherin switch in Schwann cell is unclear. Using a neuron-Schwann cell co-culture system, here we show that E-cadherin expression is induced by components on the axonal membrane. We also show that the axonal effect is mediated through cAMP-dependent protein kinase A (cAMP-PKA) activation in the Schwann cell: (1) inhibition of cAMP-PKA blocks axon-induced E-cadherin expression and (2) cAMP elevation in the Schwann cell is sufficient to induce E-cadherin expression. In addition, cAMP-dependent E-cadherin expression is promoted by contact between adjacent Schwann cell membranes, suggesting its role in autotypic junction formation during myelination. Furthermore, cAMP-induced E-cadherin expression is accompanied by suppression of N-cadherin expression. Therefore, we propose that axon-dependent activation of cAMP-PKA serves as a signal that promotes cadherin switch during postnatal development of Schwann cells.

An automated mapping method for Nissl-stained mouse brain histologic sections.

BACKGROUND: Histologic evaluation of the central nervous system is often a critical endpoint in in vivo efficacy studies, and is considered the essential component of neurotoxicity assessment in safety studies. Automated image analysis is a powerful tool that can radically reduce the workload associated with evaluating brain histologic sections. NEW METHOD: We developed an automated brain mapping method that identifies neuroanatomic structures in mouse histologic coronal brain sections. The method utilizes the publicly available Allen Brain Atlas to map brain regions on digitized Nissl-stained sections. RESULTS: The method's accuracy was first assessed by comparing the mapping results to structure delineations from the Franklin and Paxinos (FP) mouse brain atlas. Brain regions mapped from FP Nissl-stained sections and calculated volumes were similar to structure delineations and volumes derived from corresponding FP illustrations. We subsequently applied our method to mouse brain sections from an in vivo study where the hippocampus was the structure of interest. Nissl-stained sections were mapped and hippocampal boundaries transferred to adjacent immunohistochemically stained sections. Optical density quantification results were comparable to those from time-consuming, manually drawn hippocampal delineations on the IHC-stained sections. COMPARISON WITH EXISTING METHODS: Compared to other published methods, our method requires less manual input, and has been validated comprehensively using a secondary atlas, as well as manually annotated brain IHC sections from 68 study mice. CONCLUSIONS: We propose that our automated brain mapping method enables greater efficiency and consistency in mouse neuropathologic assessments.

Enhanced Neuronal Survival and Neurite Outgrowth Triggered by Novel Small Organic Compounds Mimicking the LewisX Glycan.

Glycosylation fine-tunes signal transduction of adhesion molecules during neural development and supports synaptic plasticity and repair after injury in the adult nervous system. One abundantly expressed neural glycan is LewisX (LeX). Although it is known that its expression starts at the formation of the neural tube during the second embryonic week in the mouse and peaks during the first postnatal week, its functional relevance is only rudimentarily understood. To gain better insights into the functions of this glycan, we identified small organic compounds that mimic structurally and functionally this glycan glycosidically linked to several neural adhesion molecules. Mimetic compounds were identified by competitive enzyme-linked immunosorbent assay (ELISA) using the LeX-specific monoclonal antibodies L5 and SSEA-1 for screening a library of small organic molecules. In this assay, antibody binding to substrate-coated LeX glycomimetic peptide is measured in the presence of compounds, allowing identification of molecules that inhibit antibody binding and thereby mimic LeX. Gossypol, orlistat, ursolic acid, folic acid, and tosufloxacin inhibited antibody binding in a concentration-dependent manner. With the aim to functionally characterize the molecular consequences of the compounds' actions, we here present evidence that, at nM concentrations, the mimetic compounds enhance neurite outgrowth and promote neuronal survival of cultured mouse cerebellar granule cells via, notably, distinct signal transduction pathways. These findings raise hopes that these LeX mimetics will be powerful tools for further studying the functions of LeX and its effects in acute and chronic nervous system disease models. It is worth mentioning in this context that the LeX compounds investigated in the present study have been clinically approved for different therapies.

Sizes and Sufficient Quantities of MSC Microspheres for Intrathecal Injection to Modulate Inflammation in Spinal Cord Injury.

Microencapsulation of mesenchymal stem cells (MSC) in alginate facilitates cell delivery, localization and survival, and modulates inflammation in vivo. However, we found that delivery of the widely used ~0.5 mm diameter encapsulated MSC (eMSC) by intrathecal injection into spinal cord injury (SCI) rats was highly variable. Injections of smaller (~0.2 mm) diameter eMSC into the lumbar spine were much more reproducible and they increased the anti-inflammatory macrophage response around the SCI site. We now report that injection of small eMSC >2 cm caudal from the rat SCI improved locomotion and myelin preservation 8 weeks after rat SCI versus control injections. Because preparation of sufficient quantities of small eMSC for larger studies was not feasible and injection of the large eMSC is problematic, we have developed a procedure to prepare medium-sized eMSC (~0.35 mm diameter) that can be delivered more reproducibly into the lumbar rat spine. The number of MSC incorporated/capsule in the medium sized capsules was ~5-fold greater than that in small capsules and the total yield of eMSC was ~20-fold higher than that for the small capsules. Assays with all three sizes of eMSC capsules showed that they inhibited TNF-α secretion from activated macrophages in co-cultures, suggesting no major difference in their anti-inflammatory activity in vitro. The in vivo activity of the medium-sized eMSC was tested after injecting them into the lumbar spine 1 day after SCI. Histological analyses 1 week later showed that eMSC reduced levels of activated macrophages measured by IB4 staining and increased white matter sparing in similar regions adjacent to the SCI site. The combined results indicate that ~0.35 mm diameter eMSC reduced macrophage inflammation in regions where white matter was preserved during critical early phases after SCI. These techniques enable preparation of eMSC in sufficient quantities to perform pre-clinical SCI studies with much larger numbers of subjects that will provide functional analyses of several critical parameters in rodent models for CNS inflammatory injury.

Expression and regulation of integrin receptors in human trophoblast cells: role of estradiol and cytokines.

Embryo implantation and placentation are dynamic cellular events that require not only synchrony between the maternal environment and the embryo, but also complex cell-cell communication amongst the implanting blastocyst and the receptive endometrium through integrins, a large family of proteins involved in the attachment, migration, invasion and control of cellular functions. Integrins display dynamic temporal and spatial patterns of expression by the trophoblast cells during early pregnancy in humans. However, the precise mechanism of embryo implantation and the modulation of the integrin receptors during blastocyst attachment and further implantation remain elusive in the humans. The present study elucidates the expression and hormonal modulation of fibronectin, vitronectin and laminin integrin receptors by estradiol and IL-1alpha in human trophoblast cells. Human first trimester trophoblast cells showed the induction of the classical estrogen receptor (ER)-alpha by its own ligand, estradiol. Treatment with either estradiol or IL-1alpha induced the expressions of alpha4, alpha5, alpha6 and alpha(v) integrin receptor subunits at both the mRNA and protein levels, while expression of beta1 remained unaltered. Furthermore, estradiol upregulated the expression of IL-1alpha, thereby suggesting the possibility that estrogen may either directly or via the proinflammatory cytokine induces the expression of the cell surface integrin receptors. The findings delineate the role of hormones and the cytokines in modulating the adhesiveness and attachment of the trophoblast cells. This may reflect the in vivo scenario where the implanting embryo is surrounded by a hormone-cytokine rich uterine microenvironment that may precisely regulate the expression of integrins and thereby facilitate implantation.

Juvenile and adult olfactory ensheathing cells bundle and myelinate dorsal root ganglion axons in culture.

Olfactory ensheathing cells (OEC), which normally associate closely with but do not myelinate axons in situ, myelinate axons in the adult mammalian spinal cord. They are of clinical interest as candidate cells for autologous transplantation but the ability of OEC to myelinate axons in vitro has been controversial. To clarify this issue, we isolated OEC from olfactory bulbs (OB) of juvenile and adult rats expressing GFP and analyzed their ability to myelinate axons. Using a well-defined assay for myelination of dorsal root ganglia (DRG) axons in culture, we found that OEC from juvenile pups associated with and then myelinated DRG axons. OEC assembled into bundles with the axons by 1week and required more than a week before myelination on axons was detected. In contrast, rat Schwann cells did not bundle axons and they formed P0(+) and MBP(+) myelin segments after as little as 1week. Most of the OEC in culture exhibited staining for calponin, a marker that was not found on Schwann cells in culture, whereas in both OEC and Schwann cell populations nearly all cells were positive for p75NTR and GFAP. These results confirm previous reports showing only subtle immunological differences between Schwann cells and OEC. Besides differences in the rate of myelination, we detected two additional functional differences in the interactions of OEC and Schwann cells with DRG axons. First, the diameter of OEC generated myelin was greater than for Schwann cell myelin on DRG axons. Second, OEC but not Schwann cells myelinated DRG axons in the absence of vitamin C. OEC isolated from adult OB were also found to bundle and myelinate DRG axons but the latter occurred only after incubation times of at least 3weeks. The results indicate that adult OEC require longer incubation times than juvenile OEC to myelinate axons and suggest that patterns of myelination by OEC and Schwann cells are distinguishable at least on axons in vitro. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

Neurofascin interactions play a critical role in clustering sodium channels, ankyrin G and beta IV spectrin at peripheral nodes of Ranvier.

The Ig cell adhesion molecules (CAM) neurofascin (NF) and Nr-CAM are localized at developing nodes of Ranvier in peripheral myelinated axons prior to clustering of Na+ channels. Different isoforms of NF are expressed on neurons and glia, and NF binding on both cells has been suggested to play roles in node and paranode formation. To clarify the role of NF further, we analyzed effects of NF-Fc fusion proteins in Schwann cell-DRG neuron myelinating cocultures. NF-Fc significantly inhibited nodal clustering of Na+ channels, ankyrin G, and betaIV spectrin, and modestly reduced Caspr clustering at paranodal junctions; it did not significantly affect lengths or numbers of myelin-positive segments, axon initial segments, or accumulations of phosphorylated-ERM proteins in Schwann cell nodal microvilli. NF-Fc binds to Schwann cells but little or no binding to DRG neurons was detected. The results suggest a critical early role for axonal NF in clustering of Na+ channels at nodes of Ranvier via interactions with receptors on Schwann cells.

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation.

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.

Expression of pro-inflammatory cytokines in mouse blastocysts during implantation: modulation by steroid hormones.

PROBLEM: Expression and hormonal regulation of pro-inflammatory cytokines and their role in blastocyst activation and implantation is poorly known. The present study is aimed at analysing the expression and hormonal modulation of two pro-inflammatory cytokines [interleukin-1alpha (IL-1alpha) and IL-6] in mouse blastocysts during implantation. METHOD OF STUDY: Blastocyst-uterine interactions are inhibited by progesterone during implantation and subsequent treatment with oestrogen triggers events that allow implantation to begin. Using this delayed implantation mouse model, dormant and activated blastocysts were recovered from mice treated with progesterone alone and progesterone plus oestrogen therapy, respectively. Expression of IL-1alpha and IL-6 messenger RNA (mRNA) was analysed in normal, dormant and activated blastocysts by in situ hybridization using specific labelled sense and antisense RNA probes, and the protein expression of the same was analysed by immunocytochemistry. RESULTS: In situ hybridization revealed IL-1alpha and IL-6 mRNA localization in normal, dormant and activated blastocysts and a differential expression was observed in relation to the exposure to progesterone and oestrogen. There was less expression in the dormant blastocysts as compared with the normal and activated ones, and the pattern was similar for both cytokines. Immunocytochemistry also revealed a similar pattern of protein expression to that of the mRNA expression for both the cytokines. CONCLUSIONS: Using a delayed implantation model, we show that mouse blastocysts express both IL-1alpha and IL-6 mRNA as well as their respective proteins. Both mRNA and the protein levels of IL-1alpha and IL-6 seem to be hormonally modulated in mouse blastocysts during implantation.