Astrocytes lure CXCR2-expressing CD4+ T cells to gray matter via TAK1-mediated chemokine production in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system driven by peripheral immune cell infiltration and glial activation. The pathological hallmark of MS is demyelination, and mounting evidence suggests neuronal damage in gray matter is a major contributor to disease irreversibility. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Here, we used a modified EAE mouse model (Type-B EAE) that displays severe neuronal damage to investigate the interplay between peripheral immune cells and glial cells in the event of neuronal damage. We show that CD4+ T cells migrate to the spinal cord gray matter, preferentially to ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobilized and interacted with neurons, which are behaviors associated with detrimental effects to normal neuronal function. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Further, astrocyte-targeted deletion of TAK1 inhibited production of CXCR2 ligands such as CXCL1 in gray matter, successfully prevented T cell migration into spinal cord gray matter, and averted neuronal damage and motor dysfunction in Type-B EAE mice. This study identifies astrocyte chemokine production as a requisite for the invasion of CD4+T cell into the gray matter to induce neuronal damage.

Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic debilitating immune-mediated disease of the central nervous system (CNS) driven by demyelination and gray matter neurodegeneration. We previously reported an experimental autoimmune encephalomyelitis (EAE) MS mouse model with elevated serum CXCL1 that developed severe and prolonged neuron damage. Our findings suggested that CXCR2 signaling may be important in neuronal damage, thus implicating neutrophils, which express CXCR2 in abundance, as a potential cell type involved. The goals of this study were to determine if CXCR2 signaling in neutrophils mediate neuronal damage and to identify potential mechanisms of damage. METHODS: EAE was induced in wild-type control and neutrophil-specific Cxcr2 knockout (Cxcr2 cKO) mice by repeated high-dose injections of heat-killed Mycobacterium tuberculosis and MOG35-55 peptide. Mice were examined daily for motor deficit. Serum CXCL1 level was determined at different time points throughout disease development. Neuronal morphology in Golgi-Cox stained lumbar spinal cord ventral horn was assessed using recently developed confocal reflection super-resolution technique. Immune cells from CNS and lymphoid organs were quantified by flow cytometry. CNS-derived neutrophils were co-cultured with neuronal crest cells and neuronal cell death was measured. Neutrophils isolated from lymphoid organs were examined for expression of reactive oxygen species (ROS) and ROS-related genes. Thioglycolate-activated neutrophils were isolated, treated with recombinant CXCL1, and measured for ROS production. RESULTS: Cxcr2 cKO mice had less severe disease symptoms at peak and late phase when compared to control mice with similar levels of CNS-infiltrating neutrophils and other immune cells despite high levels of circulating CXCL1. Additionally, Cxcr2 cKO mice had significantly reduced CNS neuronal damage in the ventral horn of the spinal cord. Neutrophils isolated from control EAE mice induced vast neuronal cell death in vitro when compared with neutrophils isolated from Cxcr2 cKO EAE mice. Neutrophils isolated from control EAE mice, but not Cxcr2 cKO mice, exhibited elevated ROS generation, in addition to heightened Ncf1 and Il1b transcription. Furthermore, recombinant CXCL1 was sufficient to significantly increase neutrophils ROS production. CONCLUSIONS: CXCR2 signal in neutrophils is critical in triggering CNS neuronal damage via ROS generation, which leads to prolonged EAE disease. These findings emphasize that CXCR2 signaling in neutrophils may be a viable target for therapeutic intervention against CNS neuronal damage.

Anatomy Nights: An international public engagement event increases audience knowledge of brain anatomy.

Anatomy Nights is an international public engagement event created to bring anatomy and anatomists back to public spaces with the goal of increasing the public's understanding of their own anatomy by comparison with non-human tissues. The event consists of a 30-minute mini-lecture on the anatomy of a specific anatomical organ followed by a dissection of animal tissues to demonstrate the same organ anatomy. Before and after the lecture and dissection, participants complete research surveys designed to assess prior knowledge and knowledge gained as a result of participation in the event, respectively. This study reports the results of Anatomy Nights brain events held at four different venues in the UK and USA in 2018 and 2019. Two general questions were asked of the data: 1) Do participant post-event test scores differ from pre-event scores; and 2) Are there differences in participant scores based on location, educational background, and career. We addressed these questions using a combination of generalized linear models (R's glm function; R version 4.1.0 [R Core Team, 2014]) that assumed a binomial distribution and implemented a logit link function, as well as likelihood estimates to compare models. Survey data from 91 participants indicate that scores improve on post-event tests compared to pre-event tests, and these results hold irrespective of location, educational background, and career. In the pre-event tests, participants performed well on naming structures with an English name (frontal lobe and brainstem), and showed signs of improvement on other anatomical names in the post-test. Despite this improvement in knowledge, we found no evidence that participation in Anatomy Nights improved participants' ability to apply this knowledge to neuroanatomical contexts (e.g., stroke).

Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles.

Fluorescence correlation spectroscopy (FCS) monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused to the C-terminus of the enhanced green fluorescent protein (EGFP). The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs). The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

Improved gene transfection efficacy and cytocompatibility of multifunctional polyamidoamine-cross-linked hyaluronan particles.

We describe a multi-functional, cationic hyaluronic acid (HA)-based gene carrier with improved transfection over non-cross-linked HA, and negligible cytotoxicity. Cationized particles are developed by cross-linking HA chain carboxyl groups with polyamidoamine amine termini to produce well segregated particles of 350-400 nm with a surface charge density of +2 mV, compared with -35 mV for non-cationized particles. A tethered antibody fragment retains ligand binding for cell targeting. Cationized and antibody-linked particles complex plasmid DNA efficiently and the cationized particles successfully deliver reporter genes to bovine intervertebral disk cells as an intervertebral disk regeneration model.

Isolation and characterisation of a recombinant antibody fragment that binds NCAM1-expressing intervertebral disc cells.

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.