Vesicular HMGB1 release from neurons stressed with spreading depolarization enables confined inflammatory signaling to astrocytes.

The role of high mobility group box 1 (HMGB1) in inflammation is well characterized in the immune system and in response to tissue injury. More recently, HMGB1 was also shown to initiate an "inflammatory signaling cascade" in the brain parenchyma after a mild and brief disturbance, such as cortical spreading depolarization (CSD), leading to headache. Despite substantial evidence implying a role for inflammatory signaling in prevalent neuropsychiatric disorders such as migraine and depression, how HMGB1 is released from healthy neurons and how inflammatory signaling is initiated in the absence of apparent cell injury are not well characterized. We triggered a single cortical spreading depolarization by optogenetic stimulation or pinprick in naïve Swiss albino or transgenic Thy1-ChR2-YFP and hGFAP-GFP adult mice. We evaluated HMGB1 release in brain tissue sections prepared from these mice by immunofluorescent labeling and immunoelectron microscopy. EzColocalization and Costes thresholding algorithms were used to assess the colocalization of small extracellular vesicles (sEVs) carrying HMGB1 with astrocyte or microglia processes. sEVs were also isolated from the brain after CSD, and neuron-derived sEVs were captured by CD171 (L1CAM). sEVs were characterized with flow cytometry, scanning electron microscopy, nanoparticle tracking analysis, and Western blotting. We found that HMGB1 is released mainly within sEVs from the soma of stressed neurons, which are taken up by surrounding astrocyte processes. This creates conditions for selective communication between neurons and astrocytes bypassing microglia, as evidenced by activation of the proinflammatory transcription factor NF-ĸB p65 in astrocytes but not in microglia. Transmission immunoelectron microscopy data illustrated that HMGB1 was incorporated into sEVs through endosomal mechanisms. In conclusion, proinflammatory mediators released within sEVs can induce cell-specific inflammatory signaling in the brain without activating transmembrane receptors on other cells and causing overt inflammation.

Supramolecular presentation of bioinstructive peptides on soft multilayered nanobiomaterials stimulates neurite outgrowth.

Peptide amphiphiles (PAs) have emerged as effective molecular building blocks for creating self-assembling nanobiomaterials for multiple biomedical applications. Herein, we report a straightforward approach to assemble soft bioinstructive platforms to recreate the native neural extracellular matrix (ECM) aiming for neuronal regeneration based on the electrostatic-driven supramolecular presentation of laminin-derived IKVAV-containing self-assembling PA (IKVAV-PA) on biocompatible multilayered nanoassemblies. Spectroscopic and microscopic techniques show that the co-assembly of positively charged low-molecular-weight IKVAV-PA with oppositely charged high-molecular-weight hyaluronic acid (HA) triggers the formation of ordered ß-sheet structures denoting a one-dimensional nanofibrous network. The successful functionalization of poly(L-lysine)/HA layer-by-layer nanofilms with an outer positively charged layer of self-assembling IKVAV-PA is demonstrated by the quartz crystal microbalance with dissipation monitoring and the nanofibrous morphological properties revealed by atomic force microscopy. The bioactive ECM-mimetic supramolecular nanofilms promote the enhancement of primary neuronal cells' adhesion, viability, and morphology when compared to the PA without the IKVAV sequence and PA-free biopolymeric multilayered nanofilms, and stimulate neurite outgrowth. The nanofilms hold great promise as bioinstructive platforms for enabling the assembly of customized and robust multicomponent supramolecular biomaterials for neural tissue regeneration.

Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity.

BACKGROUND: Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. METHODS: We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. RESULTS: Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. CONCLUSION: In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Overview of extracellular vesicle characterization techniques and introduction to combined reflectance and fluorescence confocal microscopy to distinguish extracellular vesicle subpopulations.

Extracellular vesicles (EVs) are nanoparticles (30 to 1000 nm in diameter) surrounded by a lipid-bilayer which carry bioactive molecules between local and distal cells and participate in intercellular communication. Because of their small size and heterogenous nature they are challenging to characterize. Here, we discuss commonly used techniques that have been employed to yield information about EV size, concentration, mechanical properties, and protein content. These include dynamic light scattering, nanoparticle tracking analysis, flow cytometry, transmission electron microscopy, atomic force microscopy, western blotting, and optical methods including super-resolution microscopy. We also introduce an innovative technique for EV characterization which involves immobilizing EVs on a microscope slide before staining them with antibodies targeting EV proteins, then using the reflectance mode on a confocal microscope to locate the EV plane. By then switching to the microscope's fluorescence mode, immunostained EVs bearing specific proteins can be identified and the heterogeneity of an EV preparation can be determined. This approach does not require specialist equipment beyond the confocal microscopes that are available in many cell biology laboratories, and because of this, it could become a complementary approach alongside the aforementioned techniques to identify molecular heterogeneity in an EV preparation before subsequent analysis requiring specialist apparatus.

Doxycycline alleviates acute traumatic brain injury by suppressing neuroinflammation and apoptosis in a mouse model.

Traumatic brain injury (TBI) is one of the significant causes of death among young people worldwide. Doxycycline (DOX), an antibiotic with anti-inflammatory effects, has not been used as a therapeutic agent to modify the inflammatory response after the traumatic brain injury. In this study, intraperitoneal administration of DOX reduced significantly the acute inflammatory markers like IL-6 and CD3, microglial migration to the damaged area marked with Iba-1, and neuronal apoptosis assessed with TUNEL assay at 72 h after the trauma. The low dose, 10 mg/kg of DOX had a dominant anti-inflammatory effect; while the high dose, 100 mg/kg of DOX, was more effective in decreasing neuronal apoptosis. In early hours after the head trauma, use of a low dose (10 mg/kg) of DOX for decreasing the acute form of inflammation followed by a high dose (100 mg/kg) for the anti-apoptotic effects particularly in severe head traumas, would be a promising approach to alleviate the brain injury.

Neuroactive Peptide Nanofibers for Regeneration of Spinal Cord after Injury.

The highly complex nature of spinal cord injuries (SCIs) requires design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Promising SCI treatments use biomaterial scaffolds, which provide bioactive cues to the cells in order to trigger neural regeneration in the spinal cord. In this work, the use of peptide nanofibers is demonstrated, presenting protein binding and cellular adhesion epitopes in a rat model of SCI. The self-assembling peptide molecules are designed to form nanofibers, which display heparan sulfate mimetic and laminin mimetic epitopes to the cells in the spinal cord. These neuroactive nanofibers are found to support adhesion and viability of dorsal root ganglion neurons as well as neurite outgrowth in vitro and enhance tissue integrity after 6 weeks of injury in vivo. Treatment with the peptide nanofiber scaffolds also show significant behavioral improvement. These results demonstrate that it is possible to facilitate regeneration especially in the white matter of the spinal cord, which is usually damaged during the accidents using bioactive 3D nanostructures displaying high densities of laminin and heparan sulfate-mimetic epitopes on their surfaces.