Protein synthesis inhibition promotes nitric oxide generation and activation of CGKII-dependent downstream signaling pathways in the retina.

Nitric oxide is an important neuromodulator in the CNS, and its production within neurons is modulated by NMDA receptors and requires a fine-tuned availability of L-arginine. We have previously shown that globally inhibiting protein synthesis mobilizes intracellular L-arginine "pools" in retinal neurons, which concomitantly enhances neuronal nitric oxide synthase-mediated nitric oxide production. Activation of NMDA receptors also induces local inhibition of protein synthesis and L-arginine intracellular accumulation through calcium influx and stimulation of eucariotic elongation factor type 2 kinase. We hypothesized that protein synthesis inhibition might also increase intracellular L-arginine availability to induce nitric oxide-dependent activation of downstream signaling pathways. Here we show that nitric oxide produced by inhibiting protein synthesis (using cycloheximide or anisomycin) is readily coupled to AKT activation in a soluble guanylyl cyclase and cGKII-dependent manner. Knockdown of cGKII prevents cycloheximide or anisomycin-induced AKT activation and its nuclear accumulation. Moreover, in retinas from cGKII knockout mice, cycloheximide was unable to enhance AKT phosphorylation. Indeed, cycloheximide also produces an increase of ERK phosphorylation which is abrogated by a nitric oxide synthase inhibitor. In summary, we show that inhibition of protein synthesis is a previously unanticipated driving force for nitric oxide generation and activation of downstream signaling pathways including AKT and ERK in cultured retinal cells. These results may be important for the regulation of synaptic signaling and neuronal development by NMDA receptors as well as for solving conflicting data observed when using protein synthesis inhibitors for studying neuronal survival during development as well in behavior and memory studies.

cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response.

Sucrose ingestion induces rapid AMPA receptor trafficking.

The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.