Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.

Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients.

Endothelial Von Willebrand factor promotes blood-brain barrier flexibility and provides protection from hypoxia and seizures in mice.

OBJECTIVE: Aberrant blood-brain barrier (BBB) permeability is a hallmark pathology of many central nervous system diseases. von Willebrand factor (VWF) is stored in endothelial Weibel-Palade bodies from where it is released on activation into plasma and basement membrane. The role of VWF in endothelial homeostasis is unclear. The goal of this study was to assess the role of VWF in disease models associated with increased BBB permeability. APPROACH AND RESULTS: We did not find any differences in BBB permeability to Evans blue dye at baseline between wild-type and VWF(-/-) animals. We next used 2 models presenting with increased BBB permeability, hypoxia/reoxygenation and pilocarpine-induced status epilepticus, to assess the response of VWF(-/-) animals. In both models, VWF(-/-) mice maintained a tighter BBB than wild-type mice. VWF(-/-) mice fared worse in both conditions, with ≈ 100% of VWF(-/-) mice dying within 120 minutes after pilocarpine administration, whereas >80% of wild-type animals survived. Investigation into the status of tight junction proteins revealed that VWF(-/-) mice expressed more claudin-5 at baseline. In vitro work confirmed that the presence of subendothelial VWF is inhibitory to claudin-5 expression. CONCLUSIONS: VWF deficiency confers partial preservation of BBB integrity after hypoxia/reoxygenation and seizures. Surprisingly, this decrease in BBB permeability did not result in protection of animals because they demonstrated more severe pathology in both models compared with wild-type animals. These data suggest that a rigid BBB is detrimental (to the organism) during certain disease states and that VWF release may provide desired flexibility under stress.

Assessing multiple endpoints of atrazine ingestion on gravid Northern Watersnakes (Nerodia sipedon) and their offspring.

Ecotoxicological studies that focus on a single endpoint might not accurately and completely represent the true ecological effects of a contaminant. Exposure to atrazine, a widely used herbicide, disrupts endocrine function and sexual development in amphibians, but studies involving live-bearing reptiles are lacking. This study tracks several effects of atrazine ingestion from female Northern Watersnakes (Nerodia sipedon) to their offspring exposed in utero. Twenty-five gravid N. sipedon were fed fish dosed with one of the four levels of atrazine (0, 2, 20, or 200 ppb) twice weekly for the entirety of their gestation period. Endpoints for the mothers included blood estradiol levels measured weekly and survival more than 3 months. Endpoints for the offspring included morphometrics, clutch sex ratio, stillbirth, and asymmetry of dorsal scales and jaw length. Through these multiple endpoints, we show that atrazine ingestion can disrupt estradiol production in mothers, increase the likelihood of mortality from infection, alter clutch sex ratio, cause a higher proportion of stillborn offspring, and affect scale symmetry. We emphasize the need for additional research involving other reptile species using multiple endpoints to determine the full range of impacts of contaminant exposure.

Occupational-like organophosphate exposure disrupts microglia and accelerates deficits in a rat model of Alzheimer's disease.

Occupational exposure to organophosphate pesticides, such as chlorpyrifos (CPF), increases the risk of Alzheimer's disease (AD), though the mechanism is unclear. To investigate this, we subjected 4-month-old male and female wild-type (WT) and TgF344-AD rats, a transgenic AD model, to an occupational CPF exposure paradigm that recapitulates biomarkers and behavioral impairments experienced by agricultural workers. Subsequent cognition and neuropathology were analyzed over the next 20 months. CPF exposure caused chronic microglial dysregulation and accelerated neurodegeneration in both males and females. The effect on neurodegeneration was more severe in males, and was also associated with accelerated cognitive impairment. Females did not exhibit accelerated cognitive impairment after CPF exposure, and amyloid deposition and tauopathy were unchanged in both males and females. Microglial dysregulation may mediate the increased risk of AD associated with occupational organophosphate exposure, and future therapies to preserve or restore normal microglia might help prevent AD in genetically vulnerable individuals exposed to CPF or other disease-accelerating environmental agents.

Pharmacological activation of the nuclear receptor REV-ERB reverses cognitive deficits and reduces amyloid-ß burden in a mouse model of Alzheimer's disease.

Alzheimer's disease currently lacks treatment options that effectively reverse the biological/anatomical pathology and cognitive deficits associated with the disease. Loss of function of the nuclear receptor REV-ERB is associated with reduced cognitive function in mouse models. The effect of enhanced REV-ERB activity on cognitive function has not been examined. In this study, we tested the hypothesis that enhanced REV-ERB function may enhance cognitive function in a model of Alzheimer's disease. We utilized the REV-ERB agonist SR9009 to pharmacologically activate the activity of REV-ERB in the SAMP8 mouse model of Alzheimer's disease. SR9009 reversed cognitive dysfunction of an aged SAMP8 mouse in several behavioral assays including novel object recognition, T-maze foot shock avoidance, and lever press operant conditioning task assessments. SR9009 treatment reduced amyloid-ß 1-40 and 1-42 levels in the cortex, which is consistent with improved cognitive function. Furthermore, SR9009 treatment led to increased hippocampal PSD-95, cortical synaptophysin expression and the number of synapses suggesting improvement in synaptic function. We conclude that REV-ERB is a potential target for treatment of Alzheimer's disease.

(-)-P7C3-S243 Protects a Rat Model of Alzheimer's Disease From Neuropsychiatric Deficits and Neurodegeneration Without Altering Amyloid Deposition or Reactive Glia.

BACKGROUND: In addition to cognitive deficits, Alzheimer's disease (AD) is associated with other neuropsychiatric symptoms, including severe depression. Indeed, depression often precedes cognitive deficits in patients with AD. Unfortunately, the field has seen only minimal therapeutic advances, underscoring the critical need for new treatments. P7C3 aminopropyl carbazoles promote neuronal survival by enhancing nicotinamide adenine dinucleotide flux in injured neurons. Neuroprotection with P7C3 compounds has been demonstrated in preclinical models of neurodegeneration by virtue of promoting neuronal survival independently of early disease-specific pathology, resulting in protection from cognitive deficits and depressive-like behavior. We hypothesize that P7C3 compounds might be uniquely applicable to patients with AD, given the comorbid presentation of depression and cognitive deficits. METHODS: Aging male and female wild-type and TgF344-AD rats, a well-characterized preclinical AD model, were administered (-)-P7C3-S243 daily for 9 and 18 months, beginning at 6 months of age. Behavioral phenotypes related to cognition and depression were assessed at 15 and 24 months, and brain pathology and biochemistry were assessed at 24 months. RESULTS: (-)-P7C3-S243 safely protected aging male and female wild-type and TgF344-AD rats from cognitive deficits and depressive-like behavior. Depressive-like behavior occurred earlier than cognitive deficits in TgF344-AD rats, consistent with AD in many patients. Treatment with (-)-P7C3-S243 blocked neurodegeneration in TgF344-AD rats, without altering amyloid deposition or indicators of neuroinflammation. CONCLUSIONS: Neuronal cell death-specific treatment approaches, such as P7C3 compounds, may represent a new treatment approach for patients experiencing the combination of cognitive deficits and depression associated with AD.

Neurotoxicity in Preclinical Models of Occupational Exposure to Organophosphorus Compounds.

Organophosphorus (OPs) compounds are widely used as insecticides, plasticizers, and fuel additives. These compounds potently inhibit acetylcholinesterase (AChE), the enzyme that inactivates acetylcholine at neuronal synapses, and acute exposure to high OP levels can cause cholinergic crisis in humans and animals. Evidence further suggests that repeated exposure to lower OP levels insufficient to cause cholinergic crisis, frequently encountered in the occupational setting, also pose serious risks to people. For example, multiple epidemiological studies have identified associations between occupational OP exposure and neurodegenerative disease, psychiatric illness, and sensorimotor deficits. Rigorous scientific investigation of the basic science mechanisms underlying these epidemiological findings requires valid preclinical models in which tightly-regulated exposure paradigms can be correlated with neurotoxicity. Here, we review the experimental models of occupational OP exposure currently used in the field. We found that animal studies simulating occupational OP exposures do indeed show evidence of neurotoxicity, and that utilization of these models is helping illuminate the mechanisms underlying OP-induced neurological sequelae. Still, further work is necessary to evaluate exposure levels, protection methods, and treatment strategies, which taken together could serve to modify guidelines for improving workplace conditions globally.

Acute Axonal Degeneration Drives Development of Cognitive, Motor, and Visual Deficits after Blast-Mediated Traumatic Brain Injury in Mice.

Axonal degeneration is a prominent feature of many forms of neurodegeneration, and also an early event in blast-mediated traumatic brain injury (TBI), the signature injury of soldiers in Iraq and Afghanistan. It is not known, however, whether this axonal degeneration is what drives development of subsequent neurologic deficits after the injury. The Wallerian degeneration slow strain (WldS) of mice is resistant to some forms of axonal degeneration because of a triplicated fusion gene encoding the first 70 amino acids of Ufd2a, a ubiquitin-chain assembly factor, that is linked to the complete coding sequence of nicotinamide mononucleotide adenylyltransferase 1 (NMAT1). Here, we demonstrate that resistance of WldS mice to axonal degeneration after blast-mediated TBI is associated with preserved function in hippocampal-dependent spatial memory, cerebellar-dependent motor balance, and retinal and optic nerve-dependent visual function. Thus, early axonal degeneration is likely a critical driver of subsequent neurobehavioral complications of blast-mediated TBI. Future therapeutic strategies targeted specifically at mitigating axonal degeneration may provide a uniquely beneficial approach to treating patients suffering from the effects of blast-mediated TBI.

P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

Lack of tryptophan hydroxylase-1 in mice results in gait abnormalities.

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (-/-) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system.

ADAMTS13 exerts a thrombolytic effect in microcirculation.

Recombinant tissue plasminogen activator (r-tPA) is the drug of choice for thrombolysis, but it is associated with a significant risk of bleeding and is not always successful. By cleaving von Willebrand factor (VWF), the metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats-13) down-regulates thrombus formation in injured vessels. We investigated whether recombinant ADAMTS13 (r-ADAMTS13) induces thrombolysis in vivo in mice. Thrombosis was produced by ferric chloride-induced (FeCl(3)) injury in the venules of a dorsal skinfold chamber. Phosphate-buffered saline (PBS, vehicle), r-tPA or r-ADAMTS13, supplemented with hirudin (to stop on-going thrombin generation), was directly applied onto the occluded vessel, and thrombus dissolution was evaluated by intravital microscopy. The incidence of blood flow restoration significantly increased 30 minutes (min) after r-ADAMTS13 vs. PBS treatment (60% vs. 0%, p<0.05) and 60 min after r-tPA treatment (75% vs. 17%, p<0.05). Both r-tPA and r-ADAMTS13 significantly reduced thrombus size 60 min after their superfusion (53.2% and 62.3% of the initial thrombus size, p<0.05 and p<0.01, respectively). Bleeding occurred in all r-tPA-treated chambers, while it was absent in mice treated with r-ADAMTS13 or PBS. We observed that, similar to r-tPA, r-ADAMTS13 can dissolve occlusive thrombi induced by FeCl(3) injury in venules. In contrast to r-tPA, the in vivo thrombolytic effect of ADAMTS13 was not associated with any signs of haemorrhage. ADAMTS13 could represent a new therapeutic option for thrombolysis.