NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.

Here we provide evidence in support of an inherent role for Arpc1b, a component of the Arp2/3 complex, in regulation of mitosis and demonstrate that its depletion inhibits Aurora A activation at the centrosome and impairs the ability of mammalian cells to enter mitosis. We discovered that Arpc1b colocalizes with gamma-tubulin at centrosomes and stimulates Aurora A activity. Aurora A phosphorylates Arpc1b on threonine 21, and expression of Arpc1b but not a nonphosphorylatable Arpc1b mutant in mammalian cells leads to Aurora A kinase activation and abnormal centrosome amplification in a Pak1-independent manner. Together, these findings reveal a new function for Arpc1b in centrosomal homeostasis. Arpc1b is both a physiological activator and substrate of Aurora A kinase and these interactions help to maintain mitotic integrity in mammalian cells.

Pak1 plays an important role in various cellular processes, including cell motility, polarity, survival and proliferation. To date, its role in atherogenesis has not been explored. Here we report the effect of Pak1 on atherogenesis using atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) mice as a model. Disruption of Pak1 in ApoE(-/-) mice results in reduced plaque burden, significantly attenuates circulating IL-6 and MCP-1 levels, limits the expression of adhesion molecules and diminishes the macrophage content in the aortic root of ApoE(-/-) mice. We also observed reduced oxidized LDL uptake and increased cholesterol efflux by macrophages and smooth muscle cells of ApoE(-/-):Pak1(-/-) mice as compared with ApoE(-/-) mice. In addition, we detect increased Pak1 phosphorylation in human atherosclerotic arteries, suggesting its role in human atherogenesis. Altogether, these results identify Pak1 as an important factor in the initiation and progression of atherogenesis.

Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.

Based on the hypothesis that brain plaques and tangles can affect cortical function in Alzheimer's disease (AD), we investigated functional responses in an AD rat model (called the Samaritan Alzheimer's rat achieved by ventricular infusion of amyloid peptide) and age-matched healthy control. High-field functional magnetic resonance imaging (fMRI) and extracellular neural activity measurements were applied to characterize sensory-evoked responses. Electrical stimulation of the forepaw led to BOLD and neural responses in the contralateral somatosensory cortex and thalamus. In AD brain we noted much smaller BOLD activation patterns in the somatosensory cortex (i.e., about 50% less activated voxels compared to normal brain). While magnitudes of BOLD and neural responses in the cerebral cortex were markedly attenuated in AD rats compared to normal rats (by about 50%), the dynamic coupling between the BOLD and neural responses in the cerebral cortex, as assessed by transfer function analysis, remained unaltered between the groups. However thalamic BOLD and neural responses were unaltered in AD brain compared to controls. Thus cortical responses in the AD model were indeed diminished compared to controls, but the thalamic responses in the AD and control rats were quite similar. Therefore these results suggest that Alzheimer's disease may affect cortical function more than subcortical function, which may have implications for interpreting altered human brain functional responses in fMRI studies of Alzheimer's disease.

Mitochondrial Ca(2+) uptake, central to neural metabolism and function, is diminished in aging whereas enhanced after acute/sub-acute traumatic brain injury. To develop relevant translational models for these neuropathologies, we determined the impact of perturbed mitochondrial Ca(2+) uptake capacities on intrinsic brain activity using clinically relevant markers. From a multi-compartment estimate of probable baseline Ca(2+) ranges in the brain, we hypothesized that reduced or enhanced mitochondrial Ca(2+) uptake capacity would decrease or increase spontaneous neuronal activity respectively. As resting state fMRI-BOLD fluctuations and stimulus-evoked BOLD responses have similar physiological origins [1] and stimulus-evoked neuronal and hemodynamic responses are modulated by mitochondrial Ca(2+) uptake capacity [2], [3] respectively, we tested our hypothesis by measuring hemodynamic fluctuations and spontaneous neuronal activities during normal and altered mitochondrial functional states. Mitochondrial Ca(2+) uptake capacity was perturbed by pharmacologically inhibiting or enhancing the mitochondrial Ca(2+) uniporter (mCU) activity. Neuronal electrical activity and cerebral blood flow (CBF) fluctuations were measured simultaneously and integrated with fMRI-BOLD fluctuations at 11.7T. mCU inhibition reduced spontaneous neuronal activity and the resting state functional connectivity (RSFC), whereas mCU enhancement increased spontaneous neuronal activity but reduced RSFC. We conclude that increased or decreased mitochondrial Ca(2+) uptake capacities lead to diminished resting state modes of brain functional connectivity.

Developmental nicotine exposure causes persistent changes in cortical neuron morphology and in behavior. We used microarray screening to identify master transcriptional or epigenetic regulators mediating these effects of nicotine and discovered increases in Ash2l mRNA, encoding a component of a histone methyltransferase complex. We therefore examined genome-wide changes in trimethylation of histone H3 on Lys4 (H3K4me3), a mark induced by the Ash2l complex associated with increased gene transcription. A large proportion of regulated promoter sites were involved in synapse maintenance. We found that Mef2c interacts with Ash2l and mediates changes in H3K4me3. Knockdown of Ash2l or Mef2c abolished nicotine-mediated alterations of dendritic complexity in vitro and in vivo, and attenuated nicotine-dependent changes in passive avoidance behavior. In contrast, overexpression mimicked nicotine-mediated alterations of neuronal structure and passive avoidance behavior. These studies identify Ash2l as a target induced by nicotinic stimulation that couples developmental nicotine exposure to changes in brain epigenetic marks, neuronal structure and behavior.

Focal cortical dysplasia (FCD), a local malformation of cortical development, is the most common cause of pharmacoresistant epilepsy associated with life-long neurocognitive impairments. It remains unclear whether neuronal misplacement is required for seizure activity. Here we show that dyslamination and white matter heterotopia are not necessary for seizure generation in a murine model of type II FCDs. These experimental FCDs generated by increasing mTOR activity in layer 2/3 neurons of the medial prefrontal cortex are associated with tonic-clonic seizures and a normal survival rate. Preventing all FCD-related defects, including neuronal misplacement and dysmorphogenesis, with rapamycin treatments from birth eliminates seizures, but seizures recur after rapamycin withdrawal. In addition, bypassing neuronal misplacement and heterotopia using inducible vectors do not prevent seizure occurrence. Collectively, data obtained using our new experimental FCD-associated epilepsy suggest that life-long treatment to reduce neuronal dysmorphogenesis is required to suppress seizures in individuals with FCD.

The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak1(-/-)) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak1(-/-) and wild type mice. Pak1(-/-) mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak1(-/-) mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness.

The postnatal subventricular zone (SVZ) contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A(2) receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF) significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.

The human cerebral cortex is distinguished by its large size and abundant gyrification, or folding. However, the evolutionary mechanisms that drive cortical size and structure are unknown. Although genes that are essential for cortical developmental expansion have been identified from the genetics of human primary microcephaly (a disorder associated with reduced brain size and intellectual disability) 1 , studies of these genes in mice, which have a smooth cortex that is one thousand times smaller than the cortex of humans, have provided limited insight. Mutations in abnormal spindle-like microcephaly-associated (ASPM), the most common recessive microcephaly gene, reduce cortical volume by at least 50% in humans2-4, but have little effect on the brains of mice5-9; this probably reflects evolutionarily divergent functions of ASPM10,11. Here we used genome editing to create a germline knockout of Aspm in the ferret (Mustela putorius furo), a species with a larger, gyrified cortex and greater neural progenitor cell diversity12-14 than mice, and closer protein sequence homology to the human ASPM protein. Aspm knockout ferrets exhibit severe microcephaly (25-40% decreases in brain weight), reflecting reduced cortical surface area without significant change in cortical thickness, as has been found in human patients3,4, suggesting that loss of 'cortical units' has occurred. The cortex of fetal Aspm knockout ferrets displays a very large premature displacement of ventricular radial glial cells to the outer subventricular zone, where many resemble outer radial glia, a subtype of neural progenitor cells that are essentially absent in mice and have been implicated in cerebral cortical expansion in primates12-16. These data suggest an evolutionary mechanism by which ASPM regulates cortical expansion by controlling the affinity of ventricular radial glial cells for the ventricular surface, thus modulating the ratio of ventricular radial glial cells, the most undifferentiated cell type, to outer radial glia, a more differentiated progenitor.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and β-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

To establish magnetic resonance (MR)-based molecular imaging paradigms for the noninvasive monitoring of extracellular pH (pHe) as a functional surrogate biomarker for metabolic changes induced by locoregional therapy of liver cancer.

Volcanoes commonly inflate or deflate during episodes of unrest or eruption. Continuum mechanics models that assume linear elastic deformation of the Earth's crust are routinely used to invert the observed ground motions. The source(s) of deformation in such models are generally interpreted in terms of magma bodies or pathways, and thus form a basis for hazard assessment and mitigation. Using discontinuum mechanics models, we show how host-rock fracturing (i.e. non-elastic deformation) during drainage of a magma body can progressively change the shape and depth of an elastic-deformation source. We argue that this effect explains the marked spatio-temporal changes in source model attributes inferred for the March-April 2007 eruption of Piton de la Fournaise volcano, La Reunion. We find that pronounced deflation-related host-rock fracturing can: (1) yield inclined source model geometries for a horizontal magma body; (2) cause significant upward migration of an elastic-deformation source, leading to underestimation of the true magma body depth and potentially to a misinterpretation of ascending magma; and (3) at least partly explain underestimation by elastic-deformation sources of changes in sub-surface magma volume.

We synthesized manganese ferrite (MnFe2O4) nanoparticles of different sizes by varying pH during chemical co-precipitation procedure and modified their surfaces with polysaccharide chitosan (CS) to investigate characteristics of hyperthermia and magnetic resonance imaging (MRI). Structural features were analyzed by X-ray diffraction (XRD), high-resolution transmission electron microscopy (TEM), selected area diffraction (SAED) patterns, and Mössbauer spectroscopy to confirm the formation of superparamagnetic MnFe2O4 nanoparticles with a size range of 5-15 nm for pH of 9-12. The hydrodynamic sizes of nanoparticles were less than 250 nm with a polydispersity index of 0.3, whereas the zeta potentials were higher than 30 mV to ensure electrostatic repulsion for stable colloidal suspension. MRI properties at 7T demonstrated that transverse relaxation (T2) doubled as the size of CS-coated MnFe2O4 nanoparticles tripled in vitro. However, longitudinal relaxation (T1) was strongest for the smallest CS-coated MnFe2O4 nanoparticles, as revealed by in vivo positive contrast MRI angiography. Cytotoxicity assay on HeLa cells showed CS-coated MnFe2O4 nanoparticles is viable regardless of ambient pH, whereas hyperthermia studies revealed that both the maximum temperature and specific loss power obtained by alternating magnetic field exposure depended on nanoparticle size and concentration. Overall, these results reveal the exciting potential of CS-coated MnFe2O4 nanoparticles in MRI and hyperthermia studies for biomedical research.

The ε4 allele of Apolipoprotein (APOE4) is the strongest genetic risk factor for Alzheimer's disease (AD), the most common form of dementia. Cognitively normal APOE4 carriers have developed amyloid beta (Aβ) plaques and cerebrovascular, metabolic and structural deficits decades before showing the cognitive impairment. Interventions that can inhibit Aβ retention and restore the brain functions to normal would be critical to prevent AD for the asymptomatic APOE4 carriers. A major goal of the study was to identify the potential usefulness of rapamycin (Rapa), a pharmacological intervention for extending longevity, for preventing AD in the mice that express human APOE4 gene and overexpress Aβ (the E4FAD mice). Another goal of the study was to identify the potential pharmacogenetic differences in response to rapamycin between the E4FAD and E3FAD mice, the mice with human APOE ε3 allele. We used multi-modal MRI to measure in vivo cerebral blood flow (CBF), neurotransmitter levels, white matter integrity, water content, cerebrovascular reactivity (CVR) and somatosensory response; used behavioral assessments to determine cognitive function; used biochemistry assays to determine Aβ retention and blood-brain barrier (BBB) functions; and used metabolomics to identify brain metabolic changes. We found that in the E4FAD mice, rapamycin normalized bodyweight, restored CBF (especially in female), BBB activity for Aβ transport, neurotransmitter levels, neuronal integrity and free fatty acid level, and reduced Aβ retention, which were not observe in the E3FAD-Rapa mice. In contrast, E3FAD-Rapa mice had lower CVR responses, lower anxiety and reduced glycolysis in the brain, which were not seen in the E4FAD-Rapa mice. Further, rapamycin appeared to normalize lipid-associated metabolism in the E4FAD mice, while slowed overall glucose-associated metabolism in the E3FAD mice. Finally, rapamycin enhanced overall water content, water diffusion in white matter, and spatial memory in both E3FAD and E4FAD mice, but did not impact the somatosensory responses under hindpaw stimulation. Our findings indicated that rapamycin was able to restore brain functions and reduce AD risk for young, asymptomatic E4FAD mice, and there were pharmacogenetic differences between the E3FAD and E4FAD mice. As the multi-modal MRI methods used in the study are readily to be used in humans and rapamycin is FDA-approved, our results may pave a way for future clinical testing of the pharmacogenetic responses in humans with different APOE alleles, and potentially using rapamycin to prevent AD for asymptomatic APOE4 carriers.

Because the human brain consumes a disproportionate fraction of the resting body's energy, positron emission tomography (PET) measurements of absolute glucose metabolism (CMRglc) can serve as disease biomarkers. Global mean normalization (GMN) of PET data reveals disease-based differences from healthy individuals as fractional changes across regions relative to a global mean. To assess the impact of GMN applied to metabolic data, we compared CMRglc with and without GMN in healthy awake volunteers with eyes closed (i.e., control) against specific physiological/clinical states, including healthy/awake with eyes open, healthy/awake but congenitally blind, healthy/sedated with anesthetics, and patients with disorders of consciousness. Without GMN, global CMRglc alterations compared to control were detected in all conditions except in congenitally blind where regional CMRglc variations were detected in the visual cortex. However, GMN introduced regional and bidirectional CMRglc changes at smaller fractions of the quantitative delocalized changes. While global information was lost with GMN, the quantitative approach (i.e., a validated method for quantitative baseline metabolic activity without GMN) not only preserved global CMRglc alterations induced by opening eyes, sedation, and varying consciousness but also detected regional CMRglc variations in the congenitally blind. These results caution the use of GMN upon PET-measured CMRglc data in health and disease.

While functional MRI (fMRI) localizes regions of brain activation, functional MRS (fMRS) provides insights into metabolic underpinnings. Previous fMRS studies detected task-induced lactate increase using short echo-time non-edited 1H-MRS protocols, where lactate changes depended on accurate exclusion of overlapping lactate and lipid/macromolecule signals. Because long echo-time J-difference 1H-MRS detection of lactate is less susceptible to this shortcoming, we posited if J-edited fMRS protocol could reliably detect metabolic changes in the human motor cortex during a finger-tapping paradigm in relation to a reliable measure of basal lactate. Our J-edited fMRS protocol at 4T was guided by an fMRI pre-scan to determine the 1H-MRS voxel placement in the motor cortex. Because lactate and β-hydroxybutyrate (BHB) follow similar J-evolution profiles we observed both metabolites in all spectra, but only lactate showed reproducible task-induced modulation by 0.07 mM from a basal value of 0.82 mM. These J-edited fMRS results demonstrate good sensitivity and specificity for task-induced lactate modulation, suggesting that J-edited fMRS studies can be used to investigate the metabolic underpinning of human cognition by measuring lactate dynamics associated with activation and deactivation fMRI paradigms across brain regions at magnetic field lower than 7T.

Glutamate-ammonia ligase (glutamine synthetase; Glul) is enriched in astrocytes and serves as the primary enzyme for ammonia detoxification and glutamate inactivation in the brain. Loss of astroglial Glul is reported in hippocampi of epileptic patients, but the mechanism by which Glul deficiency might cause disease remains elusive. Here we created a novel mouse model by selectively deleting Glul in the hippocampus and neocortex. The Glul deficient mice were born without any apparent malformations and behaved unremarkably until postnatal week three. There were reductions in tissue levels of aspartate, glutamate, glutamine and GABA and in mRNA encoding glutamate receptor subunits GRIA1 and GRIN2A as well as in the glutamate transporter proteins EAAT1 and EAAT2. Adult Glul-deficient mice developed progressive neurodegeneration and spontaneous seizures which increased in frequency with age. Importantly, progressive astrogliosis occurred before neurodegeneration and was first noted in astrocytes along cerebral blood vessels. The responses to CO2-provocation were attenuated at four weeks of age and dilated microvessels were observed histologically in sclerotic areas of cKO. Thus, the abnormal glutamate metabolism observed in this model appeared to cause epilepsy by first inducing gliopathy and disrupting the neurovascular coupling.

Limited view tomographic reconstruction aims to reconstruct a tomographic image from a limited number of projection views arising from sparse view or limited angle acquisitions that reduce radiation dose or shorten scanning time. However, such a reconstruction suffers from severe artifacts due to the incompleteness of sinogram. To derive quality reconstruction, previous methods use UNet-like neural architectures to directly predict the full view reconstruction from limited view data; but these methods leave the deep network architecture issue largely intact and cannot guarantee the consistency between the sinogram of the reconstructed image and the acquired sinogram, leading to a non-ideal reconstruction. In this work, we propose a cascaded residual dense spatial-channel attention network consisting of residual dense spatial-channel attention networks and projection data fidelity layers. We evaluate our methods on two datasets. Our experimental results on AAPM Low Dose CT Grand Challenge datasets demonstrate that our algorithm achieves a consistent and substantial improvement over the existing neural network methods on both limited angle reconstruction and sparse view reconstruction. In addition, our experimental results on Deep Lesion datasets demonstrate that our method is able to generate high-quality reconstruction for 8 major lesion types.