Neuronal Nsun2 deficiency produces tRNA epitranscriptomic alterations and proteomic shifts impacting synaptic signaling and behavior.

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids.

Predicting difficult airways: 3-3-2 rule or 3-3 rule?

BACKGROUND: The goal of this study was to assess the value of the 3-3 rule and the 3-3-1 rule in predicting difficult airways. METHODS: The authors conducted an observational study over a 6-month period. For each consenting adult patient undergoing general anesthesia, preoperative patient characteristics and data regarding difficult airway assessments and airway outcomes were collected. The 3-3-2 rule, 3-3-1 rule and 3-3 rule were included in preoperative difficult airway assessments. The 3-3-1 rule is defined as an interincisor distance (IID) less than three fingers, a hyoid-mental distance (HMD) less than three fingers, and a hyoid-thyroid cartilage distance (HTD) less than one finger. RESULTS: Among the 732 patients who were successfully recruited in this study, 67 patients had difficult laryngoscopy (DL) (9.2 %), and 25 patients had difficult intubation (DI) (3.4 % of the total). All of the DI patients were also DL patients (25/67, 37.3 %). The AUC of the 3-3-2, 3-3, and 3-3-1 rules for predicting difficult laryngoscopy were 0.702, 0.709, and 0.631, respectively. Significant differences between the 3-3-2 and 3-3-1 rules as well as between the 3-3 and 3-3-1 rules were evident. The AUC values for the 3-3-2, 3-3, and 3-3-1 rules for predicting DI were 0.830, 0.822, and 0.725, respectively. CONCLUSIONS: The 3-3 rule and the 3-3-2 rule are similar regarding their ability to predict difficult airways. A HTD less than two fingers or one finger is not predictive of DV or DI.

Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins.

The D(2) and D(3) receptors (D(2)R and D(3)R), which are potential targets for antipsychotic drugs, have a similar structural architecture and signaling pathway. Furthermore, in some brain regions they are expressed in the same cells, suggesting that differences between the two receptors might lie in other properties such as their regulation. In this study we investigated, using COS-7 and HEK-293 cells, the mechanism underlying the intracellular trafficking of the D(2)R and D(3)R. Activation of D(2)R caused G protein-coupled receptor kinase-dependent receptor phosphorylation, a robust translocation of beta-arrestin to the cell membrane, and profound receptor internalization. The internalization of the D(2)R was dynamin-dependent, suggesting that a clathrin-coated endocytic pathway is involved. In addition, the D(2)R, upon agonist-mediated internalization, localized to intracellular compartments distinct from those utilized by the beta(2)-adrenergic receptor. However, in the case of the D(3)R, only subtle agonist-mediated receptor phosphorylation, beta-arrestin translocation to the plasma membrane, and receptor internalization were observed. Interchange of the second and third intracellular loops of the D(2)R and D(3)R reversed their phenotypes, implicating these regions in the regulatory properties of the two receptors. Our studies thus indicate that functional distinctions between the D(2)R and D(3)R may be found in their desensitization and cellular trafficking properties. The differences in their regulatory properties suggest that they have distinct physiological roles in the brain.

Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1.

PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.

Distinct roles of CaMKII and PKA in regulation of firing patterns and K(+) currents in Drosophila neurons.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the cAMP-dependent protein kinase A (PKA) cascades have been implicated in neural mechanisms underlying learning and memory as supported by mutational analyses of the two enzymes in Drosophila. While there is mounting evidence for their roles in synaptic plasticity, less attention has been directed toward their regulation of neuronal membrane excitability and spike information coding. Here we report genetic and pharmacological analyses of the roles of PKA and CaMKII in the firing patterns and underlying K(+) currents in cultured Drosophila central neurons. Genetic perturbation of the catalytic subunit of PKA (DC0) did not alter the action potential duration but disrupted the frequency coding of spike-train responses to constant current injection in a subpopulation of neurons. In contrast, selective inhibition of CaMKII by the expression of an inhibitory peptide in ala transformants prolonged the spike duration but did not affect the spike frequency coding. Enhanced membrane excitability, indicated by spontaneous bursts of spikes, was observed in CaMKII-inhibited but not in PKA-diminished neurons. In wild-type neurons, the spike train firing patterns were highly reproducible under consistent stimulus conditions. However, disruption of either of these kinase pathways led to variable firing patterns in response to identical current stimuli delivered at a low frequency. Such variability in spike duration and frequency coding may impose problems for precision in signal processing in these protein kinase learning mutants. Pharmacological analyses of mutations that affect specific K(+) channel subunits demonstrated distinct effects of PKA and CaMKII in modulation of the kinetics and amplitude of different K(+) currents. The results suggest that PKA modulates Shaker A-type currents, whereas CaMKII modulates Shal-A type currents plus delayed rectifier Shab currents. Thus differential regulation of K(+) channels may influence the signal handling capability of neurons. This study provides support for the notion that, in addition to synaptic mechanisms, modulations in spike activity patterns may represent an important mechanism for learning and memory that should be explored more fully.

Spontaneous acetylcholine secretion from developing growth cones of Drosophila central neurons in culture: effects of cAMP-pathway mutations.

We describe a novel bioassay system that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball was manipulated into contact with cultured Drosophila "giant" neurons, spontaneous synaptic current-like events were registered. These events were observed within seconds after contact and were blocked by curare and alpha-bungarotoxin, but not by TTX and Cd(2+), suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurred not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displayed a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI, which disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. We found that these mutations affected the ACh current kinetics, but not the quantal ACh packet, and that the release frequency was greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis.

Neuronal polymorphism among natural alleles of a cGMP-dependent kinase gene, foraging, in Drosophila.

Natural variation in neuronal excitability and connectivity has not been extensively studied. In Drosophila melanogaster, a naturally maintained genetic polymorphism at a cGMP-dependent protein kinase (PKG) gene, foraging (for), is associated with alternative food search strategies among the allelic variants Rover (for(R); higher PKG activity) and sitter (for(s); lower PKG activity). We examined physiological and morphological variations in nervous systems of these allelic variants isolated from natural populations. Whole-cell current clamping revealed distinct excitability patterns, with spontaneous activities and excessive evoked firing in cultured sitter, but not Rover, neurons. Voltage-clamp examination demonstrated reduced voltage-dependent K(+) currents in sitter neurons. Focal recordings from synapses at the larval neuromuscular junction demonstrated spontaneous activity and supernumerary discharges with increased transmitter release after nerve stimulation. Immunolabeling showed more diffuse motor axon terminal projections with increased ectopic nerve entry points in sitter larval muscles. The differences between the two natural alleles was enhanced in laboratory-induced mutant alleles of the for gene. The pervasive effects of the for-PKG on neuronal excitability, synaptic transmission, and nerve connectivity illustrate the magnitude of neuronal variability in Drosophila that can be attributed to a single gene. These findings establish the consequences in cellular function for natural variation in an isoform of PKG and suggest a role for natural selection in maintaining variation in neuronal properties.

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons.

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. Molecular analysis and heterologous expression have shown that K+ channel beta subunits regulate the properties of the pore-forming alpha subunits, although how they influence neuronal K+ currents and excitability remains to be explored. We studied cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta subunit. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha-dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants were indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shalpha subunits in a distinct neuronal subpopulation. Our results suggest that alterations in beta subunit regulation, rather than elimination or addition of alpha subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity.