Blood-brain barrier (BBB) dysfunction is a hallmark of neurological conditions such as multiple sclerosis (MS) and stroke. However, the molecular mechanisms underlying neurovascular dysfunction during BBB breakdown remain elusive. MicroRNAs (miRNAs) have recently emerged as key regulators of pathogenic responses, although their role in central nervous system (CNS) microvascular disorders is largely unknown. We have identified miR-155 as a critical miRNA in neuroinflammation at the BBB. miR-155 is expressed at the neurovascular unit of individuals with MS and of mice with experimental autoimmune encephalomyelitis (EAE). In mice, loss of miR-155 reduced CNS extravasation of systemic tracers, both in EAE and in an acute systemic inflammation model induced by lipopolysaccharide. In cultured human brain endothelium, miR-155 was strongly and rapidly upregulated by inflammatory cytokines. miR-155 up-regulation mimicked cytokine-induced alterations in junctional organization and permeability, whereas inhibition of endogenous miR-155 partially prevented a cytokine-induced increase in permeability. Furthermore, miR-155 modulated brain endothelial barrier function by targeting not only cell-cell complex molecules such as annexin-2 and claudin-1, but also focal adhesion components such as DOCK-1 and syntenin-1. We propose that brain endothelial miR-155 is a negative regulator of BBB function that may constitute a novel therapeutic target for CNS neuroinflammatory disorders.
We examined the importance of membrane cholesterol for the function and expression of TRPV1 (vanilloid receptor subtype 1) and P2X(3) in adult rat dorsal root ganglion (DRG) neurons. Cholesterol, an essential component of lipid rafts, was depleted using methyl beta-cyclodextrin (MCD). We found that MCD significantly reduced TRPV1-mediated capsaicin- and proton-activated currents. By contrast, inward currents activated by alpha,beta-methylene ATP (alpha,beta-meATP), a non-hydrolysable ATP analogue, were not altered. Immunoreactivity for TRPV1, but not P2X(3), in the plasma membrane was markedly reduced by MCD. A reduction of TRPV1 protein in membrane fractions was found following cholesterol depletion. Our data show that the level of cholesterol determines the activity and the amount of membrane TRPV1, suggesting that TRPV1 might be localized in cholesterol-rich microdomains in nociceptors. The differential dependence on the membrane cholesterol of TRPV1 and P2X(3) may have physiological significance in nociception during inflammation.
The actions of the neuropoietic cytokines are mediated by the gp130 receptor, which activates several signaling molecules including the transcription factor STAT3 (signal transducer and activator of transcription), which, in turn, is subject to feedback inhibition by SOCS3 (suppressor of cytokine signaling). Activation of the gp130 receptor has been implicated in axonal growth particularly during regeneration, but the specific contribution of STAT3 is the subject of conflicting reports. Measurements of SOCS3 mRNA in rat dorsal root ganglia showed a significant induction in this inhibitory molecule after peripheral nerve injury. The functions of STAT3 and SOCS3 in adult rat primary sensory neurons were investigated in vitro through transduction of lentiviruses yielding a conditionally activated STAT3, native SOCS3, or a mutant SOCS3 with dominant-negative actions. The SOCS3 construct was effective in inhibiting tyrosine phosphorylation of STAT3 in a neuroblastoma cell line and in blocking nuclear accumulation of endogenous STAT3 or of the conditionally activated STAT3 chimera in primary sensory neurons. In such neurons, transduction and activation of STAT3 enhanced neurite growth, transduction with SOCS3 reduced neurite outgrowth, and transduction with mutant SOCS3 enhanced neurite growth, at least under basal conditions. In conclusion, STAT3 signaling is beneficial to axonal growth through activating transcription of unidentified genes, and SOCS3 is detrimental to axonal growth through inhibition of STAT3 and/or other transcription factors.
Self-assembled nanofibers are ubiquitous in nature and serve as inspiration for the design of supramolecular hydrogels. A multicomponent approach offers the possibility of enhancing the tunability and functionality of this class of materials. We report on the synergistic multicomponent self-assembly involving a peptide amphiphile (PA) and a 1,3:2,4-dibenzylidene-d-sorbitol (DBS) gelator to generate hydrogels with tunable nanoscale morphology, improved stiffness, enhanced self-healing, and stability to enzymatic degradation. Using induced circular dichroism of Thioflavin T (ThT), electron microscopy, small-angle neutron scattering, and molecular dynamics approaches, we confirm that the PA undergoes self-sorting, while the DBS gelator acts as an additive modifier for the PA nanofibers. The supramolecular interactions between the PA and DBS gelators result in improved bulk properties and cytocompatibility of the two-component hydrogels as compared to those of the single-component systems. The tunable mechanical properties, self-healing ability, resistance to proteolysis, and biocompatibility of the hydrogels suggest future opportunities for the hydrogels as scaffolds for tissue engineering and drug delivery vehicles.
Pro-inflammatory cytokine-induced activation of nuclear factor, NF-κB has an important role in leukocyte adhesion to, and subsequent migration across, brain endothelial cells (BECs), which is crucial for the development of neuroinflammatory disorders such as multiple sclerosis (MS). In contrast, microRNA-146a (miR-146a) has emerged as an anti-inflammatory molecule by inhibiting NF-κB activity in various cell types, but its effect in BECs during neuroinflammation remains to be evaluated. Here, we show that miR-146a was upregulated in microvessels of MS-active lesions and the spinal cord of mice with experimental autoimmune encephalomyelitis. In vitro, TNFα and IFNγ treatment of human cerebral microvascular endothelial cells (hCMEC/D3) led to upregulation of miR-146a. Brain endothelial overexpression of miR-146a diminished, whereas knockdown of miR-146a augmented cytokine-stimulated adhesion of T cells to hCMEC/D3 cells, nuclear translocation of NF-κB, and expression of adhesion molecules in hCMEC/D3 cells. Furthermore, brain endothelial miR-146a modulates NF-κB activity upon cytokine activation through targeting two novel signaling transducers, RhoA and nuclear factor of activated T cells 5, as well as molecules previously identified, IL-1 receptor-associated kinase 1, and TNF receptor-associated factor 6. We propose brain endothelial miR-146a as an endogenous NF-κB inhibitor in BECs associated with decreased leukocyte adhesion during neuroinflammation.
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