Blood–spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice

Humans with ALS and transgenic rodents expressing ALS-associated superoxide dismutase (SOD1) mutations develop spontaneous blood–spinal cord barrier (BSCB) breakdown, causing microvascular spinal-cord lesions. The role of BSCB breakdown in ALS disease pathogenesis in humans and mice remains, however, unclear, although chronic blood–brain barrier opening has been shown to facilitate accumulation of toxic blood-derived products in the central nervous system, resulting in secondary neurodegenerative changes. By repairing the BSCB and/or removing the BSCB-derived injurious stimuli, we now identify that accumulation of blood-derived neurotoxic hemoglobin and iron in the spinal cord leads to early motor-neuron degeneration in SOD1^G93A mice at least in part through iron-dependent oxidant stress. Using spontaneous or warfarin-accelerated microvascular lesions, motor-neuron dysfunction and injury were found to be proportional to the degree of BSCB disruption at early disease stages in SOD1^G93A mice. Early treatment with an activated protein C analog restored BSCB integrity that developed from spontaneous or warfarin-accelerated microvascular lesions in SOD1^G93A mice and eliminated neurotoxic hemoglobin and iron deposits. Restoration of BSCB integrity delayed onset of motor-neuron impairment and degeneration. Early chelation of blood-derived iron and antioxidant treatment mitigated early motor-neuronal injury. Our data suggest that BSCB breakdown contributes to early motor-neuron degeneration in ALS mice and that restoring BSCB integrity during an early disease phase retards the disease process.The blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) prevent entry of toxic circulating molecules and cells into the central nervous system (CNS) (1). Amyotrophic lateral sclerosis (ALS) is the most prominent adult motor-neuron disorder resulting in progressive motor-neuron loss in the spinal cord, brainstem, and motor cortex (2). Most ALS cases are sporadic (90%) whereas 10% are familial ALS. Over twenty independent studies in postmortem human tissue and cerebrospinal fluid (CSF) sampling from living ALS patients have established that the BBB and BSCB are damaged in familial and sporadic ALS, as reviewed elsewhere (1, 3). This BBB and BSCB disruption has been shown by spinal-cord and/or motor-cortex accumulation of different plasma proteins (e.g., IgG, fibrin, thrombin), erythrocytes, erythrocyte-derived hemoglobin and iron-containing hemosiderin, elevated CSF/serum albumin ratios, and diminished expression or degradation of the BSCB tight-junction proteins (1, 3–5). Deposition of hemoglobin-derived iron within the CNS has also been shown in ALS patients (3, 6, 7). Because human postmortem studies reflect, however, end-stage disease, it has remained unclear as to which stage of disease is enhanced by BSCB disruption. Longitudinal CSF or BSCB imaging studies have yet to be performed in living ALS patients (3) to clarify whether spinal-cord vascular dysfunction contributes to early- or late-stage disease.Transgenic rodents expressing human ALS-associated Cu/Zn superoxide dismutase (SOD1) mutations that represent 20% of all familial cases also develop a spontaneous BBB/BSCB disruption (8–12) similar to vascular pathology reported in humans (1, 3–7). Mice with a chronic BBB disruption due to aberrant signal transduction between the central nervous system endothelial cells and pericytes or astrocytes and pericytes develop a chronic BBB opening accompanied by accumulation of toxic blood-derived products in the central nervous system and secondary functional and structural neuronal changes (13–15).To determine whether BSCB disruption contributes to fatal paralytic disease caused by expression of an ALS-causing mutant, we now report how perturbing the BSCB, repairing the BSCB, and/or removing the BSCB-derived injurious stimuli influence development of disease in SOD1^G93A mice that develop a spontaneous BSCB breakdown (8, 9, 12).     

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4⁺ T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8⁺ T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4⁺ T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.     

Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.     

Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension

It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.     

The Biodurability of Covering Materials for Metallic Stents in a Bile Flow Phantom

Background  

Covered biliary metal stents have been introduced for the purpose of overcoming tumor ingrowth and treatment of benign biliary stricture. The aim of this study was to evaluate the biodurability of three commercially available biliary metal stent covering materials [e-PTFE (expanded polytetrafluoroethylene), silicone, and polyurethane] in a bile flow phantom.