Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases.

Inhibition of 2-AG hydrolysis differentially regulates blood brain barrier permeability after injury.

BACKGROUND: Acute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential. METHODS: Induction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2. RESULTS: Here, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult. CONCLUSIONS: Our results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.

Passive immunotherapy targeting amyloid-ß reduces cerebral amyloid angiopathy and improves vascular reactivity.

Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-ß isoform(s) (predominantly amyloid-ß40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-ß40 selective antibody, to attenuate amyloid-ß accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-ß accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-ß biochemically. We hypothesized that the reduction in vascular amyloid-ß40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-ß40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-ß40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-ß species that may otherwise be detrimental to normal vessel function.

AMP kinase activation mitigates dopaminergic dysfunction and mitochondrial abnormalities in Drosophila models of Parkinson's disease.

Mutations in parkin and LRRK2 together account for the majority of familial Parkinson's disease (PD) cases. Interestingly, recent evidence implicates the involvement of parkin and LRRK2 in mitochondrial homeostasis. Supporting this, we show here by means of the Drosophila model system that, like parkin, LRRK2 mutations induce mitochondrial pathology in flies when expressed in their flight muscles, the toxic effects of which can be rescued by parkin coexpression. When expressed specifically in fly dopaminergic neurons, mutant LRRK2 results in the appearance of significantly enlarged mitochondria, a phenotype that can also be rescued by parkin coexpression. Importantly, we also identified in this study that epigallocatechin gallate (EGCG), a green tea-derived catechin, acts as a potent suppressor of dopaminergic and mitochondrial dysfunction in both mutant LRRK2 and parkin-null flies. Notably, the protective effects of EGCG are abolished when AMP-activated protein kinase (AMPK) is genetically inactivated, suggesting that EGCG-mediated neuroprotection requires AMPK. Consistent with this, direct pharmacological or genetic activation of AMPK reproduces EGCG's protective effects. Conversely, loss of AMPK activity exacerbates neuronal loss and associated phenotypes in parkin and LRRK mutant flies. Together, our results suggest the relevance of mitochondrial-associated pathway in LRRK2 and parkin-related pathogenesis, and that AMPK activation may represent a potential therapeutic strategy for these familial forms of PD.

Advanced practice nursing students: pilot test of a simulation scenario.

In the current educational environment, increasing numbers of advanced practice nursing (APN) students compete for decreasing numbers of clinical sites where patient safety is paramount. Clinical simulations with high-fidelity human patient simulators provide APN students opportunities to demonstrate clinical skills and judgment in a safe supportive environment. Development, implementation, and preliminary evaluation of a scenario for APN students are discussed. Faculty and student evaluations are reviewed as well as recommendations for future simulations.

Bioluminescence-Based Complementation Assay to Correlate Conformational Changes in Membrane-Bound Complexes with Enzymatic Function.

The proteomics field has undergone tremendous development with the introduction of many innovative methods for the identification and characterization of protein-protein interactions (PPIs). Sensitive and quantitative protein association-based techniques represent a versatile tool to probe the architecture of receptor complexes and receptor-ligand interactions and expand the drug discovery toolbox by facilitating high-throughput screening (HTS) approaches. These novel methodologies will be highly enabling for interrogation of structural determinants required for the activity of multimeric membrane-bound enzymes with unresolved crystal structure and for HTS assay development focused on unique characteristics of complex assembly instead of common catalytic features, thereby increasing specificity. We describe here an example of a binary luciferase reporter assay (NanoBiT®) to quantitatively assess the heterodimerization of the catalytically active NADPH oxidase 4 (NOX4) enzyme complex. The catalytic subunit NOX4 requires association with the protein p22phox for stabilization and enzymatic activity, but the precise manner by which these two membrane-bound proteins interact to facilitate hydrogen peroxide (H2O2) generation is currently unknown. The NanoBiT complementation reporter quantitatively determined the accurate, reduced, or failed complex assembly, which can then be confirmed by determining H2O2 release, protein expression, and heterodimer trafficking. Multimeric complex formation differs between NOX enzyme isoforms, facilitating isoform-specific, PPI-based drug screening in the future.

Volumetric MRI and MRS provide sensitive measures of Alzheimer's disease neuropathology in inducible Tau transgenic mice (rTg4510).

The purpose of this study was to determine if in vivo high resolution 3D MRI and localized (1)H MR spectroscopy (MRS) can detect brain findings resembling Alzheimer's disease in a transgenic mouse model of Tau pathology. Seven double transgenic rTg4510 female mice and 7 age-matched wild-type (wt) female mice were evaluated at 5 months of age. To confirm the usefulness and consistency of in vivo MRI/S, we also scanned the brains of 14 male mice (7 rTg4510 and 7 age-matched wt) at 8 months of age. Mean hippocampal and cerebral cortex volumes in the female rTg4510 mice were 26.7% and 20.6% smaller than that in the wt controls (p<0.0001), respectively. Mean hippocampal and cerebral cortex volumes in the male rTg4510 mice were 18.4% and 16.9% smaller than that in the wt controls (p<0.00005), respectively. The mean volumes of the cerebellum were not statistically different between the rTg4510 and the wt groups. MRS assessment revealed that the myo-inositol to total creatine ratios (mIns/tCr), a measure of gliosis, were significantly higher in the hippocampus of rTg4510 mice relative to wt mice (p=0.03 for the females; p=0.005 for the males). Immunohistochemistry and histology in the same animals verified previously published data showing elevation of hyperphosphorylated Tau, glial activation and cortical and hippocampal neuronal loss. This study demonstrates that in vivo MRI/S can be a non-invasive biomarker to assess brain atrophy and related biochemical changes in the rTg4510 mouse model.

Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization.

The stabilization and activation of NOX4 through its binding with p22phox are well documented; however little is known of the precise manner by which these two proteins interact. In recent years, the field of proteomics has undergone tremendous development with the introduction of many novel methods for the identification and characterization of protein-protein interactions (PPIs). To enhance our understanding of structural determinants leading to the association between NOX4 and p22phox, we developed a binary luciferase reporter assay (NanoBiT®) to quantitatively assess NOX4-p22phox heterodimerization. The complementation reporter quantitatively determines the accurate, reduced, or failed complex assembly, which can be confirmed and further interrogated by analyzing NOX4 catalytic activity (H2O2 release), protein expression, and dimer localization. This association-based PPI technique represents both a much-needed expansion of the NOX4 lead discovery tool box and a versatile method to probe the architecture of NOX and DUOX complexes in the future.

Colitis susceptibility in mice with reactive oxygen species deficiency is mediated by mucus barrier and immune defense defects.

Reactive oxygen species (ROS) generated by NADPH oxidases (NOX/DUOX) provide antimicrobial defense, redox signaling, and gut barrier maintenance. Inactivating NOX variants are associated with comorbid intestinal inflammation in chronic granulomatous disease (CGD; NOX2) and pediatric inflammatory bowel disease (IBD; NOX1); however Nox-deficient mice do not reflect human disease susceptibility. Here we assessed if a hypomorphic patient-relevant CGD mutation will increase the risk for intestinal inflammation in mice. Cyba (p22phox) mutant mice generated low intestinal ROS, while maintaining Nox4 function. The Cyba variant caused profound mucus layer disruption with bacterial penetration into crypts, dysbiosis, and a compromised innate immune response to invading microbes, leading to mortality. Approaches used in treatment-resistant CGD or pediatric IBD such as bone marrow transplantation or oral antibiotic treatment ameliorated or prevented disease in mice. The Cyba mutant mouse phenotype implicates loss of both mucus barrier and efficient innate immune defense in the pathogenesis of intestinal inflammation due to ROS deficiency, supporting a combined-hit model where a single disease variant compromises different cellular functions in interdependent compartments.

Genetic or pharmacological activation of the Drosophila PGC-1α ortholog spargel rescues the disease phenotypes of genetic models of Parkinson's disease.

Despite intensive research, the etiology of Parkinson's disease (PD) remains poorly understood and the disease remains incurable. However, compelling evidence gathered over decades of research strongly support a role for mitochondrial dysfunction in PD pathogenesis. Related to this, PGC-1α, a key regulator of mitochondrial biogenesis, has recently been proposed to be an attractive target for intervention in PD. Here, we showed that silencing of expression of the Drosophila PGC-1α ortholog spargel results in PD-related phenotypes in flies and also seem to negate the effects of AMPK activation, which we have previously demonstrated to be neuroprotective, that is, AMPK-mediated neuroprotection appears to require PGC-1α. Importantly, we further showed that genetic or pharmacological activation of the Drosophila PGC-1α ortholog spargel is sufficient to rescue the disease phenotypes of Parkin and LRRK2 genetic fly models of PD, thus supporting the proposed use of PGC-1α-related strategies for neuroprotection in PD.

Genetic disorders coupled to ROS deficiency.

Maintaining the redox balance between generation and elimination of reactive oxygen species (ROS) is critical for health. Disturbances such as continuously elevated ROS levels will result in oxidative stress and development of disease, but likewise, insufficient ROS production will be detrimental to health. Reduced or even complete loss of ROS generation originates mainly from inactivating variants in genes encoding for NADPH oxidase complexes. In particular, deficiency in phagocyte Nox2 oxidase function due to genetic variants (CYBB, CYBA, NCF1, NCF2, NCF4) has been recognized as a direct cause of chronic granulomatous disease (CGD), an inherited immune disorder. More recently, additional diseases have been linked to functionally altered variants in genes encoding for other NADPH oxidases, such as for DUOX2/DUOXA2 in congenital hypothyroidism, or for the Nox2 complex, NOX1 and DUOX2 as risk factors for inflammatory bowel disease. A comprehensive overview of novel developments in terms of Nox/Duox-deficiency disorders is presented, combined with insights gained from structure-function studies that will aid in predicting functional defects of clinical variants.

Efficacy of Vitamin D compounds to modulate estrogen receptor negative breast cancer growth and invasion.

In estrogen receptor (ER) positive breast cancer cells such as MCF-7 cells, the anti-tumor effects of 1,25(OH)(2)D(3) (1,25D(3)) may be secondary to disruption of estrogen mediated survival signals. If so, then sensitivity to 1,25D(3) mediated growth arrest could be reduced in estrogen independent breast cancer cells. The aim of these studies was to determine the effects of 1,25D(3) and EB1089 on the ER negative, invasive human breast cancer cell line SUM-159PT. 1,25D(3) and EB1089 reduced SUM-159PT cell growth subsequent to elevation of p27 and p21 levels. 1,25D(3) mediated apoptosis of SUM-159PT cells was associated with an enrichment of membrane bound bax, a redistribution of cytochome c from the mitochondria to the cytosol and PARP cleavage. 1,25D(3) and EB1089 also inhibited SUM-159PT cell invasion through an 8 microM Matrigel membrane. In pre-clinical studies, EB1089 dramatically reduced the growth of SUM-159PT xenografts in nude mice. The decreased size of tumors from EB1089 treated mice was associated with decreased proliferation and increased DNA fragmentation. Our data support the concept that Vitamin D(3) compounds trigger apoptosis by mechanisms independent of estrogen signaling. These studies indicate that Vitamin D(3) based therapeutics may be beneficial, alone or in conjunction with other agents, for the treatment of estrogen independent breast cancer.

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse.

RATIONALE: Escitalopram appears to be a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, there by curtailing the elevation of extracellular 5-hydroxytryptamine (5-HTExt), and hence anti-depressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram's inhibition here of. OBJECTIVES: Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HTExt elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HTExt elevation and/or modulates the effect of R-citalopram. METHODS: Recombinant generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying). RESULTS: We generated mice expressing either the wild-type human SERT (hSERT(WT)) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT(ALI/VFL+SI/TT)). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HTExt levels. Escitalopram-induced 5-HTExt elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site. The behavioral effects of the clinically relevant escitalopram dose were small and tended to be enhanced by R-citalopram co-administration. CONCLUSIONS: We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram.

High throughput object-based image analysis of ß-amyloid plaques in human and transgenic mouse brain.

Advances in imaging technology have enabled automated approaches for quantitative image analysis. In this study, a high content object based image analysis method was developed for quantification of ß-amyloid (Aß) plaques in postmortem brains of Alzheimer's disease (AD) subjects and in transgenic mice over overexpressing Aß. Digital images acquired from immunohistochemically stained sections of the superior frontal gyrus were analyzed for Aß plaque burden using a Definiens object-based segmentation approach. Blinded evaluation of Aß stained sections from AD and aged matched human subjects accurately identified AD cases with one exception. Brains from transgenic mice overexpressing Aß (PS1APP mice) were also evaluated by our Definiens object based image analysis approach. We observed an age-dependent increase in the amount of Aß plaque load that we quantified in both the hippocampus and cortex. From the contralateral hemisphere, we measured the amount of Aß in brain homogenates biochemically and observed a significant correlation between our biochemical measurements and those that we measured by our object based Definiens system in the hippocampus. Assessment of Aß plaque load in PS1APP mice using a manual segmentation technique (Image-Pro Plus) confirmed the results of our object-based image analysis approach. Image acquisition and analysis of 32 stained human slides and 100 mouse slides were executed in 8 h and 22 h, respectively supporting the relatively high throughput features of the Definiens platform. The data show that digital imaging combined with object based image analysis is a reliable and efficient approach to quantifying Aß plaques in human and mouse brain.

Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis.

BACKGROUND: The inbred mouse strain BTBR T+ tf/J (BTBR) exhibits behavioral deficits that mimic the core deficits of autism. Neuroanatomically, the BTBR strain is also characterized by a complete absence of the corpus callosum. The goal of this study was to identify novel molecular and cellular changes in the BTBR mouse, focusing on neuronal, synaptic, glial and plasticity markers in the limbic system as a model for identifying putative molecular and cellular substrates associated with autistic behaviors. METHODS: Forebrains of 8 to 10-week-old male BTBR and age-matched C57Bl/6J control mice were evaluated by immunohistochemistry using free-floating and paraffin embedded sections. Twenty antibodies directed against antigens specific to neurons, synapses and glia were used. Nissl, Timm and acetylcholinesterase (AchE) stains were performed to assess cytoarchitecture, mossy fibers and cholinergic fiber density, respectively. In the hippocampus, quantitative stereological estimates for the mitotic marker bromodeoxyuridine (BrdU) were performed to determine hippocampal progenitor proliferation, survival and differentiation, and brain-derived neurotrophic factor (BDNF) mRNA was quantified by in situ hybridization. Quantitative image analysis was performed for NG2, doublecortin (DCX), NeuroD, GAD67 and Poly-Sialic Acid Neural Cell Adhesion Molecule (PSA-NCAM). RESULTS: In midline structures including the region of the absent corpus callosum of BTBR mice, the myelin markers 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP) were reduced, and the oligodendrocyte precursor NG2 was increased. MBP and CNPase were expressed in small ectopic white matter bundles within the cingulate cortex. Microglia and astrocytes showed no evidence of gliosis, yet orientations of glial fibers were altered in specific white-matter areas. In the hippocampus, evidence of reduced neurogenesis included significant reductions in the number of doublecortin, PSA-NCAM and NeuroD immunoreactive cells in the subgranular zone of the dentate gyrus, and a marked reduction in the number of 5-bromo-2'-deoxyuridine (BrdU) positive progenitors. Furthermore, a significant and profound reduction in BDNF mRNA was seen in the BTBR dentate gyrus. No significant differences were seen in the expression of AchE, mossy fiber synapses or immunoreactivities of microtubule-associated protein MAP2, parvalbumin and glutamate decarboxylase GAD65 or GAD67 isoforms. CONCLUSIONS: We documented modest and selective alterations in glia, neurons and synapses in BTBR forebrain, along with reduced neurogenesis in the adult hippocampus. Of all markers examined, the most distinctive changes were seen in the neurodevelopmental proteins NG2, PSA-NCAM, NeuroD and DCX. Our results are consistent with aberrant development of the nervous system in BTBR mice, and may reveal novel substrates to link callosal abnormalities and autistic behaviors. The changes that we observed in the BTBR mice suggest potential novel therapeutic strategies for intervention in autism spectrum disorders.