Lesions and neurologic disability in inflammatory CNS diseases such as multiple sclerosis (MS) result from the translocation of leukocytes and humoral factors from the vasculature, first across the ...endothelial blood-brain barrier (BBB) and then across the astrocytic glia limitans (GL). Factors secreted by reactive astrocytes open the BBB by disrupting endothelial tight junctions (TJs), but the mechanisms that control access across the GL are unknown. Here, we report that in inflammatory lesions, a second barrier composed of reactive astrocyte TJs of claudin 1 (CLDN1), CLDN4, and junctional adhesion molecule A (JAM-A) subunits is induced at the GL. In a human coculture model, CLDN4-deficient astrocytes were unable to control lymphocyte segregation. In models of CNS inflammation and MS, mice with astrocyte-specific Cldn4 deletion displayed exacerbated leukocyte and humoral infiltration, neuropathology, motor disability, and mortality. These findings identify a second inducible barrier to CNS entry at the GL. This barrier may be therapeutically targetable in inflammatory CNS disease.
Breakdown of the blood-brain barrier (BBB) is an early and significant event in CNS inflammation. Astrocyte-derived VEGF-A has been implicated in this response, but the underlying mechanisms remain ...unresolved. Here, we identify the endothelial transmembrane tight junction proteins claudin-5 (CLN-5) and occludin (OCLN) as targets of VEGF-A action. Down-regulation of CLN-5 and OCLN accompanied up-regulation of VEGF-A and correlated with BBB breakdown in experimental autoimmune encephalomyelitis, an animal model of CNS inflammatory disease. In cultures of brain microvascular endothelial cells, VEGF-A specifically down-regulated CLN-5 and OCLN protein and mRNA. In mouse cerebral cortex, microinjection of VEGF-A disrupted CLN-5 and OCLN and induced loss of barrier function. Importantly, functional studies revealed that expression of recombinant CLN-5 protected brain microvascular endothelial cell cultures from a VEGF-induced increase in paracellular permeability, whereas recombinant OCLN expressed under the same promoter was not protective. Previous studies have shown CLN-5 to be a key determinant of trans-endothelial resistance at the BBB. Our findings suggest that its down-regulation by VEGF-A constitutes a significant mechanism in BBB breakdown.
Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The ...common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 (DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only 'healthy' retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.
Mounting evidence suggests neuroinflammation is a key process in glaucoma, yet the precise roles are not known. Understanding these complex processes, which may also be a key in other common ...neurodegenerations such as Alzheimer's disease, will lead to targeted therapeutics for a disease that affects as many as 80 million people worldwide. Here, we define neuroinflammation as any immune-relevant response by a variety of cell types including astrocytes, microglia, and peripherally derived cells occurring in the optic nerve head and/or retina. In this review article, we first discuss clinical evidence for neuroinflammation in glaucoma and define neuroinflammation in glaucoma. We then review the inflammatory pathways that have been associated with glaucoma. Finally, we set out key research directions that we believe will greatly advance our understanding of the role of neuroinflammation in glaucoma. This review arose from a discussion of neuroinflammation in glaucoma at the 2015 meeting of The Lasker/IRRF Initiative for Innovation in Vision Science. This manuscript sets out to summarize one of these sessions; “Inflammation and Glaucomatous Neurodegeneration”, as well as to review the current state of the literature surrounding neuroinflammation in glaucoma.
•We describe the clinical relevance of inflammation in glaucoma.•We define the sites of neuroinflammation in glaucoma.•We discuss the roles of different cells and pathways in glaucoma.•We discuss suggested 5 year goals as set about by the participants of the 2015 Lasker/IRRF Initiative session “Inflammation and Glaucomatous Neurodegeneration”.
Neurofibromatosis 1 (NF1) is a comparatively common autosomal dominant disorder. However, relatively few studies have assessed lifetime risk; and information about the effect of NF1 on mortality ...remains uncertain. NF1 patients were identified using The North West regional family Genetic Register, which covers the 4.1 million people living in North West England, including the regions of Greater Manchester, Cheshire and Cumbria. Data relating to tumours and malignancies were obtained from The North West Cancer Intelligence Service. Death data for the general North West population were obtained from the Office of National Statistics. We identified 1186 individuals with NF1, of whom 1023 lived within the strict regional boundaries (constituting a region of North West England bound by The Pennines to the east and Irish Sea to the west, but excluding the conurbation of Liverpool (Merseyside) and the Wirral peninsula) and 131 had died. MPNST and glioma were found to be the two most common causes of reduced life expectancy among NF1 patients. In Kaplan-Meier analyses the median survival for NF1 patients was shown to be 71.5 years, with women living ∼7.4 years longer than men. On average both men and women lived ∼8 years less than their counterparts in the general population. Reduction in life expectancy for NF1 patients was found to be much lower (8 years) than the previously estimated 15-year decrease. Limitations relating to the underreporting of NF1 on death certificates were once again highlighted and should be considered in future investigations.
Glaucoma is a complex, multifactorial disease characterised by the loss of retinal ganglion cells and their axons leading to a decrease in visual function. The earliest events that damage retinal ...ganglion cells in glaucoma are currently unknown. Retinal ganglion cell death appears to be compartmentalised, with soma, dendrite and axon changes potentially occurring through different mechanisms. There is mounting evidence from other neurodegenerative diseases suggesting that neuronal dendrites undergo a prolonged period of atrophy, including the pruning of synapses, prior to cell loss. In addition, recent evidence has shown the role of the complement cascade in synaptic pruning in glaucoma and other diseases.
Using a genetic (DBA/2J mouse) and an inducible (rat microbead) model of glaucoma we first demonstrate that there is loss of retinal ganglion cell synapses and dendrites at time points that precede axon or soma loss. We next determine the role of complement component 1 (C1) in early synaptic loss and dendritic atrophy during glaucoma. Using a genetic knockout of C1qa (D2.C1qa (-/-) mouse) or pharmacological inhibition of C1 (in the rat bead model) we show that inhibition of C1 is sufficient to preserve dendritic and synaptic architecture.
This study further supports assessing the potential for complement-modulating therapeutics for the prevention of retinal ganglion cell degeneration in glaucoma.
Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the ...liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.
In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ...ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an astrocyte-derived permeability factor, and suggest TYMP and VEGFA together promote blood-brain barrier breakdown.
Oligodendrocytes derive from progenitors (OPCs) through the interplay of epigenomic and transcriptional events. By integrating high-resolution methylomics, RNA-sequencing, and multiple transgenic ...lines, this study defines the role of DNMT1 in developmental myelination. We detected hypermethylation of genes related to cell cycle and neurogenesis during differentiation of OPCs, yet genetic ablation of Dnmt1 resulted in inefficient OPC expansion and severe hypomyelination associated with ataxia and tremors in mice. This phenotype was not caused by lineage switch or massive apoptosis but was characterized by a profound defect of differentiation associated with changes in exon-skipping and intron-retention splicing events and by the activation of an endoplasmic reticulum stress response. Therefore, loss of Dnmt1 in OPCs is not sufficient to induce a lineage switch but acts as an important determinant of the coordination between RNA splicing and protein synthesis necessary for myelin formation.
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•Oligodendrocyte-lineage-specific analysis of DNA methylome and transcriptome•No fate switch induced by Dnmt1 ablation in early oligodendrocyte progenitors•DNMT1-dependent regulation of differentiation-specific alternative splicing events•Impaired myelination in Dnmt1 mutants characterized by aberrant ER stress response
Moyon et al. combine high-resolution methylomics, RNA sequencing, and analysis of multiple transgenic lines to define a critical role for DNA methylation in developmental myelination. Ablation of Dnmt1 decreases proliferation yet minimally impacts progenitors while impairing the late stages of differentiation, which require alternative splicing.
In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier ...(BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A-induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.
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