Brief exposure to obesogenic diet disrupts brain dopamine networks.

OBJECTIVE: We have previously demonstrated that insulin signaling, through the downstream signaling kinase Akt, is a potent modulator of dopamine transporter (DAT) activity, which fine-tunes dopamine (DA) signaling at the synapse. This suggests a mechanism by which impaired neuronal insulin receptor signaling, a hallmark of diet-induced obesity, may contribute to impaired DA transmission. We tested whether a short-term (two-week) obesogenic high-fat (HF) diet could reduce striatal Akt activity, a marker of central insulin, receptor signaling and blunt striatal and dopaminergic network responsiveness to amphetamine (AMPH). METHODS: We examined the effects of a two-week HF diet on striatal DAT activity in rats, using AMPH as a probe in a functional magnetic resonance imaging (fMRI) assay, and mapped the disruption in AMPH-evoked functional connectivity between key dopaminergic targets and their projection areas using correlation and permutation analyses. We used phosphorylation of the Akt substrate GSK3α in striatal extracts as a measure of insulin receptor signaling. Finally, we confirmed the impact of HF diet on striatal DA D2 receptor (D2R) availability using [18F]fallypride positron emission tomography (PET). RESULTS: We found that rats fed a HF diet for only two weeks have reductions in striatal Akt activity, a marker of decreased striatal insulin receptor signaling and blunted striatal responsiveness to AMPH. HF feeding also reduced interactions between elements of the mesolimbic (nucleus accumbens-anterior cingulate) and sensorimotor circuits (caudate/putamen-thalamus-sensorimotor cortex) implicated in hedonic feeding. D2R availability was reduced in HF-fed animals. CONCLUSION: These studies support the hypothesis that central insulin signaling and dopaminergic neurotransmission are already altered after short-term HF feeding. Because AMPH induces DA efflux and brain activation, in large part via DAT, these findings suggest that blunted central nervous system insulin receptor signaling through a HF diet can impair DA homeostasis, thereby disrupting cognitive and reward circuitry involved in the regulation of hedonic feeding.

SPECIFIC MOLECULAR RECOGNITION AS A STRATEGY TO DELINEATE TUMOR MARGIN USING TOPICALLY APPLIED FLUORESCENCE EMBEDDED NANOPARTICLES.

The Thomsen-Friedenreich (TF) antigen is a tumor-associated antigen consistently expressed on the apical surface of epithelial-based cancer cells, including pancreatic cancer. In this work, we report the development of multimodal imaging probe, the tripolymer fluorescent nanospheres, whose surface was fabricated with peanut agglutinin (PNA) moieties as TF molecular recognition molecules. Here, we demonstrate that the probe is able to detect TF antigen in human pancreatic cancer tissues and differentiate from normal tissue. What is most noteworthy regarding the probe is its ability to visualize tumor margins defined by epithelial TF antigen expression. Further, in vivo preclinical studies using an orthotopic mouse model of pancreatic cancer suggest the potential use of the nanospheres for laparoscopic imaging of pancreatic cancer tumor margins to enhance surgical resection and improve clinical outcomes.

Improved proliferation of antigen-specific cytolytic T lymphocytes using a multimodal nanovaccine.

The present study investigated the immunoenhancing property of our newly designed nanovaccine, that is, its ability to induce antigen-specific immunity. This study also evaluated the synergistic effect of a novel compound PBS-44, an α-galactosylceramide analog, in boosting the immune response induced by our nanovaccine. The nanovaccine was prepared by encapsulating ovalbumin (ova) and an adjuvant within the poly(lactic-co-glycolic acid) nanoparticles. Quantitative analysis of our study data showed that the encapsulated vaccine was physically and biologically stable; the core content of our nanovaccine was found to be released steadily and slowly, and nearly 90% of the core content was slowly released over the course of 25 days. The in vivo immunization studies exhibited that the nanovaccine induced stronger and longer immune responses compared to its soluble counterpart. Similarly, intranasal inhalation of the nanovaccine induced more robust antigen-specific CD8+ T cell response than intraperitoneal injection of nanovaccine.

mTORC2/rictor signaling disrupts dopamine-dependent behaviors via defects in striatal dopamine neurotransmission.

Disrupted neuronal protein kinase B (Akt) signaling has been associated with dopamine (DA)-related neuropsychiatric disorders, including schizophrenia, a devastating mental illness. We hypothesize that proper DA neurotransmission is therefore dependent upon intact neuronal Akt function. Akt is activated by phosphorylation of two key residues: Thr308 and Ser473. Blunted Akt phosphorylation at Ser473 (pAkt-473) has been observed in lymphocytes and postmortem brains of schizophrenia patients, and psychosis-prone normal individuals. Mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multiprotein complex that is responsible for phosphorylation of Akt at Ser473 (pAkt-473). We demonstrate that mice with disrupted mTORC2 signaling in brain exhibit altered striatal DA-dependent behaviors, such as increased basal locomotion, stereotypic counts, and exaggerated response to the psychomotor effects of amphetamine (AMPH). Combining in vivo and ex vivo pharmacological, electrophysiological, and biochemical techniques, we demonstrate that the changes in striatal DA neurotransmission and associated behaviors are caused, at least in part, by elevated D2 DA receptor (D2R) expression and upregulated ERK1/2 activation. Haloperidol, a typical antipsychotic and D2R blocker, reduced AMPH hypersensitivity and elevated pERK1/2 to the levels of control animals. By viral gene delivery, we downregulated mTORC2 solely in the dorsal striatum of adult wild-type mice, demonstrating that striatal mTORC2 regulates AMPH-stimulated behaviors. Our findings implicate mTORC2 signaling as a novel pathway regulating striatal DA tone and D2R signaling.

Fluorescence-based endoscopic imaging of Thomsen-Friedenreich antigen to improve early detection of colorectal cancer.

Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.

Rictor/mTORC2 facilitates central regulation of energy and glucose homeostasis.

Insulin signaling in the central nervous system (CNS) regulates energy balance and peripheral glucose homeostasis. Rictor is a key regulatory/structural subunit of the mTORC2 complex and is required for hydrophobic motif site phosphorylation of Akt at serine 473. To examine the contribution of neuronal Rictor/mTORC2 signaling to CNS regulation of energy and glucose homeostasis, we utilized Cre-LoxP technology to generate mice lacking Rictor in all neurons, or in either POMC or AgRP expressing neurons. Rictor deletion in all neurons led to increased fat mass and adiposity, glucose intolerance and behavioral leptin resistance. Disrupting Rictor in POMC neurons also caused obesity and hyperphagia, fasting hyperglycemia and pronounced glucose intolerance. AgRP neuron specific deletion did not impact energy balance but led to mild glucose intolerance. Collectively, we show that Rictor/mTORC2 signaling, especially in POMC-expressing neurons, is important for central regulation of energy and glucose homeostasis.

Neuronal ablation of p-Akt at Ser473 leads to altered 5-HT1A/2A receptor function.

The serotonergic system regulates a wide range of behavior, including mood and impulsivity, and its dysregulation has been associated with mood disorders, autism spectrum disorder, and addiction. Diabetes is a risk factor for these conditions. Insulin resistance in the brain is specifically associated with susceptibility to psychostimulant abuse. Here, we examined whether phosphorylation of Akt, a key regulator of the insulin signaling pathway, controls serotonin (5-HT) signaling. To explore how impairment in Akt function regulates 5-HT homeostasis, we used a brain-specific rictor knockout (KO) mouse model of impaired neuronal phosphorylation of Akt at Ser473. Cortical 5-HT1A and 5-HT2A receptor binding was significantly elevated in rictor KO mice. Concomitant with this elevated receptor expression, the 5-HT1A receptor agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) led to an increased hypothermic response in rictor KO mice. The increased cortical 5-HT1A receptor density was associated with higher 5-HT1A receptor levels on the cortical cell surface. In contrast, rictor KO mice displayed significantly reduced head-twitch response (HTR) to the 5-HT2A/C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI), with evidence of impaired 5-HT2A/C receptor signaling. In vitro, pharmacological inhibition of Akt significantly increased 5-HT1A receptor expression and attenuated DOI-induced 5-HT2A receptor signaling, thereby lending credence to the observed in vivo cross-talk between neuronal Akt signaling and 5-HT receptor regulation. These data reveal that defective central Akt function alters 5-HT signaling as well as 5-HT-associated behaviors, demonstrating a novel role for Akt in maintaining neuronal 5-HT receptor function.

Nitric oxide in cerebral vasospasm: theories, measurement, and treatment.

In recent decades, a large body of research has focused on the role of nitric oxide (NO) in the development of cerebral vasospasm (CV) following subarachnoid hemorrhage (SAH). Literature searches were therefore conducted regarding the role of NO in cerebral vasospasm, specifically focusing on NO donors, reactive nitrogen species, and peroxynitrite in manifestation of vasospasm. Based off the assessment of available evidence, two competing theories are reviewed regarding the role of NO in vasospasm. One school of thought describes a deficiency in NO due to scavenging by hemoglobin in the cisternal space, leading to an NO signaling deficit and vasospastic collapse. A second hypothesis focuses on the dysfunction of nitric oxide synthase, an enzyme that synthesizes NO, and subsequent generation of reactive nitrogen species. Both theories have strong experimental evidence behind them and hold promise for translation into clinical practice. Furthermore, NO donors show definitive promise for preventing vasospasm at the angiographic and clinical level. However, NO augmentation may also cause systemic hypotension and worsen vasospasm due to oxidative distress. Recent evidence indicates that targeting NOS dysfunction, for example, through erythropoietin or statin administration, also shows promise at preventing vasospasm and neurotoxicity. Ultimately, the role of NO in neurovascular disease is complex. Neither of these theories is mutually exclusive, and both should be considered for future research directions and treatment strategies.

Role for mTOR signaling and neuronal activity in morphine-induced adaptations in ventral tegmental area dopamine neurons.

While the abuse of opiate drugs continues to rise, the neuroadaptations that occur with long-term drug exposure remain poorly understood. We describe here a series of chronic morphine-induced adaptations in ventral tegmental area (VTA) dopamine neurons, which are mediated via downregulation of AKT-mTORC2 (mammalian target of rapamycin complex-2). Chronic opiates decrease the size of VTA dopamine neurons in rodents, an effect seen in humans as well, and concomitantly increase the excitability of the cells but decrease dopamine output to target regions. Chronic morphine decreases mTORC2 activity, and overexpression of Rictor, a component of mTORC2, prevents morphine-induced changes in cell morphology and activity. Further, local knockout of Rictor in VTA decreases DA soma size and reduces rewarding responses to morphine, consistent with the hypothesis that these adaptations represent a mechanism of reward tolerance. Together, these findings demonstrate a novel role for AKT-mTORC2 signaling in mediating neuroadaptations to opiate drugs of abuse.

Dysregulation of the norepinephrine transporter sustains cortical hypodopaminergia and schizophrenia-like behaviors in neuronal rictor null mice.

The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder. We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE), unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of monoamine homeostasis in the cortex.

Genetic contributions to white matter architecture revealed by diffusion tensor imaging in Williams syndrome.

Little is known about genetic regulation of the development of white matter. This knowledge is critical in understanding the pathophysiology of neurodevelopmental syndromes associated with altered cognition as well as in elucidating the genetics of normal human cognition. The hemideletion of approximately 25 genes on chromosome 7q11.23 that causes Williams syndrome (WS) includes genes that regulate cytoskeletal dynamics in neurons, especially LIMK1 and CYLN2, and therefore offers the opportunity to investigate the role of these genes in the formation of white matter tracts. We used diffusion tensor imaging to demonstrate alteration in white matter fiber directionality, deviation in posterior fiber tract course, and reduced lateralization of fiber coherence in WS. These abnormalities are consistent with an alteration of the late stages of neuronal migration, define alterations of white matter structures underlying dissociable behavioral phenotypes in WS, and provide human in vivo information about genetic control of white matter tract formation.

Short course of omeprazole: a better first diagnostic approach to noncardiac chest pain than endoscopy, manometry, or 24-hour esophageal pH monitoring.

UNLABELLED: Noncardiac chest pain (NCCP) presents as a frequent diagnostic challenge, with patients tending to use a disproportionate level of health care resources. Gastroesophageal reflux disease (GERD) is the most frequent cause of NCCP. GOALS: To test the efficacy of a potent acid-suppressing agent as a diagnostic test in the evaluation of NCCP and to compare it with three commonly used tests. STUDY: Eighteen men and 24 women, aged 22 to 77 years, who presented with recurrent chest pain complaints of a noncardiac etiology, as determined by rest/stress perfusion imaging with technetium Tc99m sestamibi (MIBI), were enrolled in a prospective, double-blinded, placebo-controlled, crossover trial using high-dose omeprazole. Thirty-seven patients completed both arms of the trial. Findings were compared with those of endoscopy, manometry, and ambulatory 24-hour two-channel esophageal pH monitoring. All patients underwent initial diagnostic upper endoscopy, esophageal manometry, and 24-hour pH monitoring. Patients were then randomly assigned to either placebo or omeprazole (40 mg/d orally twice daily) for 14 days, washed out for 21 days, and then crossed over. Patient's symptoms were determined using a Visual Analogue Scale to measure the severity of chest pain before and after each period. RESULTS: Seventy-one percent of patients in the omeprazole arm reported improved chest pain, whereas only 18% in the placebo arm did. Abnormal results on manometry (20%), 24-hour pH monitoring (42%), or endoscopy with visual evidence of esophagitis (26%) were found less frequently. Combination of the three tests did not significantly increase their usefulness. In NCCP patients with GERD, as defined by positive results on a 24-hour pH test or presence of esophagitis on endoscopy, omeprazole treatment led to a response in 95% of patients, whereas 90% of GERD-positive patients treated with placebo did not respond. Of NCCP patients determined to be GERD negative, 39% responded to omeprazole. CONCLUSIONS: Omeprazole as a first diagnostic tool in the evaluation of MIBI-negative NCCP is sensitive and specific for determining the cause of NCCP. Endoscopy, manometry, and 24-hour pH monitoring were not only less sensitive in diagnosing NCCP, but they were significantly more expensive.