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

Abstract 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

Comparison of Gene-Transfer Efficiency in Human Embryonic Stem Cells

Technologies designed to allow manipulation and modification of human embryonic stem (hES) cells are numerous and vary in the complexity of their methods, efficiency, reliability, and safety. The most commonly studied and practiced of these methods include electroporation, lipofection, nucleofection, and lentiviral transduction. However, at present, it is unclear which protocol offers the most efficient and reliable method of gene transfer to hES cells. In this study, a bi-fusion construct with ubiquitin promoter driving enhanced green fluorescent protein reporter and the firefly luciferase (pUb-eGFP-Fluc) along with neomycin selection marker was used for in vitro and in vivo studies. In vitro studies examined the transfection efficiency and viability of each technique using two hES cell lines (male H1 and female H9 cells). Lentiviral transduction demonstrated the highest efficiency (H1: 25.3 ± 4.8%; H9: 22.4 ± 6.5%) with >95% cell viability. Nucleofection demonstrated transfection efficiency of 16.1 ± 3.6% (H1) and 5.8 ± 3.2% (H9). However, minimal transfection efficiency was observed with electroporation (2.1 ± 0.4% (H1) and 1.9 ± 0.3% (H9)) and lipofection (1.5 ± 0.5% (H1) and 1.3 ± 0.2% (H9); P < 0.05 vs. lentiviral transduction). Electroporation also demonstrated the highest cell death (62 ± 11% (H1) and 42 ± 10% (H9)) followed by nucleofection (25 ± 9% (H1) and 30 ± 15 (H9)). Importantly, lentiviral transduction generated a greater number of hES cell lines stably expressing the double-fusion reporter gene (hES-DF) compared to other transfection techniques. Finally, following subcutaneous transplantation into immunodeficient nude mice, the hES-eGFP-Fluc cells showed robust proliferation as determined by longitudinal bioluminescence imaging. In summary, this study demonstrates that lentiviral transduction and nucleofection are efficient, simple, and safe techniques for reliable gene transfer in hES cells. The double-fusion construct provides an attractive approach for generating stable hES cell lines and monitoring engraftment and proliferation in vitro and in vivo

Nucleofection induces non-specific changes in the metabolic activity of transfected cells

Transfection has become an everyday technique widely used for functional studies in living cells. The choice of the particular transfection method is usually determined by its efficiency and toxicity, and possible functional consequences specific to the method used are normally overlooked. We describe here that nucleofection, a method increasingly used because of its convenience and high efficiency, increases the metabolic rate of some cancer cells, which can be misleading when used as a measure of proliferation. Moreover, nucleofection can alter the subcellular expression pattern of the transfected protein. These undesired effects are independent of the transfected nucleic acid, but depend on the particular cell line used. Therefore, the interpretation of functional data using this technology requires further controls and caution

Dysregulation of the Norepinephrine Transporter Sustains Cortical Hypodopaminergia and Schizophrenia-Like Behaviors in Neuronal Rictor Null Mice

A novel animal model highlights the link between Akt dysfunction, reduced cortical dopamine function, norepinephrine transporters, and schizophrenia-like behaviors

Ligand-Dependent Conformations and Dynamics of the Serotonin 5-HT2A Receptor Determine Its Activation and Membrane-Driven Oligomerization Properties

From computational simulations of a serotonin 2A receptor (5-HT2AR) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT2AR, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization

Rapid Effects of Hearing Song on Catecholaminergic Activity in the Songbird Auditory Pathway

Catecholaminergic (CA) neurons innervate sensory areas and affect the processing of sensory signals. For example, in birds, CA fibers innervate the auditory pathway at each level, including the midbrain, thalamus, and forebrain. We have shown previously that in female European starlings, CA activity in the auditory forebrain can be enhanced by exposure to attractive male song for one week. It is not known, however, whether hearing song can initiate that activity more rapidly. Here, we exposed estrogen-primed, female white-throated sparrows to conspecific male song and looked for evidence of rapid synthesis of catecholamines in auditory areas. In one hemisphere of the brain, we used immunohistochemistry to detect the phosphorylation of tyrosine hydroxylase (TH), a rate-limiting enzyme in the CA synthetic pathway. We found that immunoreactivity for TH phosphorylated at serine 40 increased dramatically in the auditory forebrain, but not the auditory thalamus and midbrain, after 15 min of song exposure. In the other hemisphere, we used high pressure liquid chromatography to measure catecholamines and their metabolites. We found that two dopamine metabolites, dihydroxyphenylacetic acid and homovanillic acid, increased in the auditory forebrain but not the auditory midbrain after 30 min of exposure to conspecific song. Our results are consistent with the hypothesis that exposure to a behaviorally relevant auditory stimulus rapidly induces CA activity, which may play a role in auditory responses