17ß-estradiol ameliorates delirium-like phenotypes in a murine model of urinary tract infection.

Urinary tract infections (UTIs) are common and frequently precipitate delirium-like states. Advanced age coincident with the postmenopausal period is a risk factor for delirium following UTIs. We previously demonstrated a pathological role for interleukin-6 (IL-6) in mediating delirium-like phenotypes in a murine model of UTI. Estrogen has been implicated in reducing peripheral IL-6 expression, but it is unknown whether the increased susceptibility of postmenopausal females to developing delirium concomitant with UTIs reflects diminished effects of circulating estrogen. Here, we tested this hypothesis in a mouse model of UTI. Female C57BL/6J mice were oophorectomized, UTIs induced by transurethral inoculation of E. coli, and treated with 17ß-estradiol. Delirium-like behaviors were evaluated prior to and following UTI and 17ß-estradiol treatment. Compared to controls, mice treated with 17ß-estradiol had less neuronal injury, improved delirium-like behaviors, and less plasma and frontal cortex IL-6. In vitro studies further showed that 17ß-estradiol may also directly mediate neuronal protection, suggesting pleiotropic mechanisms of 17ß-estradiol-mediated neuroprotection. In summary, we demonstrate a beneficial role for 17ß-estradiol in ameliorating acute UTI-induced structural and functional delirium-like phenotypes. These findings provide pre-clinical justification for 17ß-estradiol as a therapeutic target to ameliorate delirium following UTI.

Prone positioning reduces frontal and hippocampal neuronal dysfunction in a murine model of ventilator-induced lung injury.

Prone positioning is an established treatment for severe acute lung injury conditions. Neuronal dysfunction frequently occurs with mechanical ventilation-induced acute lung injury (VILI) and clinically manifests as delirium. We previously reported a pathological role for systemic interleukin 6 (IL-6) in mediating neuronal injury. However, currently no studies have investigated the relationship between prone or supine positioning and IL-6 mediated neuronal dysfunction. Here, we hypothesize that prone positioning mitigates neuronal injury, via decreased IL-6, in a model of VILI. VILI was induced by subjecting C57BL/6J mice to high tidal volume (35 cc/kg) mechanical ventilation. Neuronal injury markers [cleaved caspase-3 (CC3), c-fos, heat shock protein 90 (Hsp90)] and inflammatory cytokines (IL-6, IL-1ß, TNF-α) were measured in the frontal cortex and hippocampus. We found statistically significantly less neuronal injury (CC3, c-Fos, Hsp90) and inflammatory cytokines (IL-6, IL-1ß, TNF-α) in the frontal cortex and hippocampus with prone compared to supine positioning (p < 0.001) despite no significant group differences in oxygen saturation or inflammatory infiltrates in the bronchoalveolar fluid (p > 0.05). Although there were no group differences in plasma IL-6 concentrations, there was significantly less cortical and hippocampal IL-6 in the prone position (p < 0.0001), indicating supine positioning may enhance brain susceptibility to systemic IL-6 during VILI via the IL-6 trans-signaling pathway. These findings call for future clinical studies to assess the relationship between prone positioning and delirium and for investigations into novel diagnostic or therapeutic paradigms to mitigate delirium by reducing expression of systemic and cerebral IL-6.

Systemic interleukin-6 inhibition ameliorates acute neuropsychiatric phenotypes in a murine model of acute lung injury.

Acute neuropsychiatric impairments occur in over 70% of patients with acute lung injury. Mechanical ventilation is a well-known precipitant of acute lung injury and is strongly associated with the development of acute delirium and anxiety phenotypes. In prior studies, we demonstrated that IL-6 mediates neuropathological changes in the frontal cortex and hippocampus of animals with mechanical ventilation-induced brain injury; however, the effect of systemic IL-6 inhibition on structural and functional acute neuropsychiatric phenotypes is not known. We hypothesized that a murine model of mechanical ventilation-induced acute lung injury (VILI) would induce neural injury to the amygdala and hippocampus, brain regions that are implicated in diverse neuropsychiatric conditions, and corresponding delirium- and anxiety-like functional impairments. Furthermore, we hypothesized that these structural and functional changes would reverse with systemic IL-6 inhibition. VILI was induced using high tidal volume (35 cc/kg) mechanical ventilation. Cleaved caspase-3 (CC3) expression was quantified as a neural injury marker and found to be significantly increased in the VILI group compared to spontaneously breathing or anesthetized and mechanically ventilated mice with 10 cc/kg tidal volume. VILI mice treated with systemic IL-6 inhibition had significantly reduced amygdalar and hippocampal CC3 expression compared to saline-treated animals and demonstrated amelioration in acute neuropsychiatric behaviors in open field, elevated plus maze, and Y-maze tests. Overall, these data provide evidence of a pathogenic role of systemic IL-6 in mediating structural and functional acute neuropsychiatric symptoms in VILI and provide preclinical justification to assess IL-6 inhibition as a potential intervention to ameliorate acute neuropsychiatric phenotypes following VILI.

Interleukin-6 mediates delirium-like phenotypes in a murine model of urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) is frequently implicated as a precipitant of delirium, which refers to an acute confusional state that is associated with high mortality, increased length of stay, and long-term cognitive decline. The pathogenesis of delirium is thought to involve cytokine-mediated neuronal dysfunction of the frontal cortex and hippocampus. We hypothesized that systemic IL-6 inhibition would mitigate delirium-like phenotypes in a mouse model of UTI. METHODS: C57/BL6 mice were randomized to either: (1) non-UTI control, (2) UTI, and (3) UTI + anti-IL-6 antibody. UTI was induced by transurethral inoculation of 1 × 108 Escherichia coli. Frontal cortex and hippocampus-mediated behaviors were evaluated using functional testing and corresponding structural changes were evaluated via quantification of neuronal cleaved caspase-3 (CC3) by immunohistochemistry and western blot. IL-6 in the brain and plasma were evaluated using immunohistochemistry, ELISA, and RT-PCR. RESULTS: Compared to non-UTI control mice, mice with UTI demonstrated significantly greater impairments in frontal and hippocampus-mediated behaviors, specifically increased thigmotaxis in Open Field (p < 0.05) and reduced spontaneous alternations in Y-maze (p < 0.01), while treatment of UTI mice with systemic anti-IL-6 fully reversed these functional impairments. These behavioral impairments correlated with frontal and hippocampal neuronal CC3 changes, with significantly increased frontal and hippocampal CC3 in UTI mice compared to non-UTI controls (p < 0.0001), and full reversal of UTI-induced CC3 neuronal changes following treatment with systemic anti-IL-6 antibody (p < 0.0001). Plasma IL-6 was significantly elevated in UTI mice compared to non-UTI controls (p < 0.01) and there were positive and significant correlations between plasma IL-6 and frontal CC3 (r2 = 0.5087/p = 0.0028) and frontal IL-6 and CC3 (r2 = 0.2653, p < 0.0001). CONCLUSIONS: These data provide evidence for a role for IL-6 in mediating delirium-like phenotypes in a mouse model of UTI. These findings provide pre-clinical justification for clinical investigations of IL-6 inhibitors to treat UTI-induced delirium.

IL-6 Inhibition Reduces Neuronal Injury in a Murine Model of Ventilator-induced Lung Injury.

Mechanical ventilation is a known risk factor for delirium, a cognitive impairment characterized by dysfunction of the frontal cortex and hippocampus. Although IL-6 is upregulated in mechanical ventilation-induced lung injury (VILI) and may contribute to delirium, it is not known whether the inhibition of systemic IL-6 mitigates delirium-relevant neuropathology. To histologically define neuropathological effects of IL-6 inhibition in an experimental VILI model, VILI was simulated in anesthetized adult mice using a 35 cc/kg tidal volume mechanical ventilation model. There were two control groups, as follow: 1) spontaneously breathing or 2) anesthetized and mechanically ventilated with 10 cc/kg tidal volume to distinguish effects of anesthesia from VILI. Two hours before inducing VILI, mice were treated with either anti-IL-6 antibody, anti-IL-6 receptor antibody, or saline. Neuronal injury, stress, and inflammation were assessed using immunohistochemistry. CC3 (cleaved caspase-3), a neuronal apoptosis marker, was significantly increased in the frontal (P < 0.001) and hippocampal (P < 0.0001) brain regions and accompanied by significant increases in c-Fos and heat shock protein-90 in the frontal cortices of VILI mice compared with control mice (P < 0.001). These findings were not related to cerebral hypoxia, and there was no evidence of irreversible neuronal death. Frontal and hippocampal neuronal CC3 were significantly reduced with anti-IL-6 antibody (P < 0.01 and P < 0.0001, respectively) and anti-IL-6 receptor antibody (P < 0.05 and P < 0.0001, respectively) compared with saline VILI mice. In summary, VILI induces potentially reversible neuronal injury and inflammation in the frontal cortex and hippocampus, which is mitigated with systemic IL-6 inhibition. These data suggest a potentially novel neuroprotective role of systemic IL-6 inhibition that justifies further investigation.

Peroxynitrite supports a metabolic reprogramming in merlin-deficient Schwann cells and promotes cell survival.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder characterized by the development of bilateral vestibular schwannomas. The NF2 gene encodes the tumor suppressor merlin, and loss of merlin activity promotes tumorigenesis and causes NF2. Cellular redox signaling has been implicated in different stages of tumor development. Among reactive nitrogen species, peroxynitrite is the most powerful oxidant produced by cells. We recently showed that peroxynitrite-mediated tyrosine nitration down-regulates mitochondrial metabolism in tumor cells. However, whether peroxynitrite supports a metabolic shift that could be exploited for therapeutic development is unknown. Here, we show that vestibular schwannomas from NF2 patients and human, merlin-deficient (MD) Schwann cells have high levels of endogenous tyrosine nitration, indicating production of peroxynitrite. Furthermore, scavenging or inhibiting peroxynitrite formation significantly and selectively decreased survival of human and mouse MD-Schwann cells. Using multiple complementary methods, we also found that merlin deficiency leads to a reprogramming of energy metabolism characterized by a peroxynitrite-dependent decrease of oxidative phosphorylation and increased glycolysis and glutaminolysis. In MD-Schwann cells, scavenging of peroxynitrite increased mitochondrial oxygen consumption and membrane potential, mediated by the up-regulation of the levels and activity of mitochondrial complex IV. This increase in mitochondrial activity correlated with a decrease in the glycolytic rate and glutamine dependence. This is the first demonstration of a peroxynitrite-dependent reprogramming of energy metabolism in tumor cells. Oxidized proteins constitute a novel target for therapeutic development not only for the treatment of NF2 schwannomas but also other tumors in which peroxynitrite plays a regulatory role.

A chemical biology approach identified PI3K as a potential therapeutic target for neurofibromatosis type 2.

Mutations in the merlin tumor suppressor gene cause Neurofibromatosis type 2 (NF2), which is a disease characterized by development of multiple benign tumors in the nervous system. The current standard of care for NF2 calls for surgical resection of the characteristic tumors, often with devastating neurological consequences. There are currently no approved non-surgical therapies for NF2. In an attempt to identify much needed targets and therapeutically active compounds for NF2 treatment, we employed a chemical biology approach using ultra-high-throughput screening. To support this goal, we created a merlin-null mouse Schwann cell (MSC) line to screen for compounds that selectively decrease their viability and proliferation. We optimized conditions for 384-well plate assays and executed a proof-of-concept screen of the Library of Pharmacologically Active Compounds. Further confirmatory and selectivity assays identified phosphatidylinositol 3-kinase (PI3K) as a potential NF2 drug target. Notably, loss of merlin function is associated with activation of the PI3K/Akt pathway in human schwannomas. We report that AS605240, a PI3K inhibitor, decreased merlin-null MSC viability in a dose-dependent manner without significantly decreasing viability of control Schwann cells. AS605240 exerted its action on merlin-null MSCs by promoting caspase-dependent apoptosis and inducing autophagy. Additional PI3K inhibitors tested also decreased viability of merlin-null MSCs in a dose-dependent manner. In summary, our chemical genomic screen and subsequent hit validation studies have identified PI3K as potential target for NF2 therapy.

Nitration of Hsp90 induces cell death.

Oxidative stress is a widely recognized cause of cell death associated with neurodegeneration, inflammation, and aging. Tyrosine nitration in these conditions has been reported extensively, but whether tyrosine nitration is a marker or plays a role in the cell-death processes was unknown. Here, we show that nitration of a single tyrosine residue on a small proportion of 90-kDa heat-shock protein (Hsp90), is sufficient to induce motor neuron death by the P2X7 receptor-dependent activation of the Fas pathway. Nitrotyrosine at position 33 or 56 stimulates a toxic gain of function that turns Hsp90 into a toxic protein. Using an antibody that recognizes the nitrated Hsp90, we found immunoreactivity in motor neurons of patients with amyotrophic lateral sclerosis, in an animal model of amyotrophic lateral sclerosis, and after experimental spinal cord injury. Our findings reveal that cell death can be triggered by nitration of a single protein and highlight nitrated Hsp90 as a potential target for the development of effective therapies for a large number of pathologies.

Measuring anisotropic cell motility on curved substrates.

Schwann cell motility was observed on laminin-coated quartz cylinders with different curvatures over an 18 hour period. A new analysis based on difference images helped to determine the minimal radius of curvature, 46 µm, which restricted motility along the cylinder axis. The migration speed, measured by calculating differences between successive images in the time series, ranged between 0.3 to 0.8 µm per minute and is similar to previously reported rates for Schwann cells. Difference images provide a rapid and simple method for the analysis of cell motility on large populations of cells.

Schwannomin/merlin promotes Schwann cell elongation and influences myelin segment length.

The Neurofibromatosis type 2 tumor suppressor, schwannomin (Sch) is a plasma membrane-cytoskeleton linking protein that regulates receptor signaling and actin dynamics. We examined Sch's role in specifying morphological changes needed for Schwann cell (SC) function in vitro. Isolated Sch-GFP-expressing SCs extended bipolar processes 82% longer than those formed by GFP-expressing cells. In contrast, SCs expressing dominant negative Sch-BBA-GFP extended bipolar processes 16% shorter than controls and 64% shorter than Sch-GFP-expressing SCs. nf2 gene inactivation caused isolated mouse SCs to transition from bipolar to multipolar cells. Live imaging revealed that SCs co-expressing Sch-GFP and dominant negative RacN17 behaved similarly in dorsal root ganglion explant cultures; they quickly aligned on axons and slowly elongated bipolar processes. In contrast, SCs expressing constitutively active RacV12 underwent continuous transitions in morphology that interfered with axon alignment. When co-cultured with neurons under myelin-promoting conditions, Sch-GFP-expressing SCs elaborated longer myelin segments than GFP-expressing SCs. In contrast, Sch-BBA-GFP-expressing SCs failed to align on or myelinate axons. Together, these results demonstrate that Sch plays an essential role in inducing and/or maintaining the SC's spindle shape and suggest that the mechanism involves Sch-dependent inhibition of Rac activity. By stabilizing the bipolar morphology, Sch promotes the alignment of SCs with axons and ultimately influences myelin segment length.