Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine-and cAMP-regulated phosphoprotein M-r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L-and T-type voltage-gated Ca2+ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine-and cAMP-regulated phosphoprotein M-r 32 000 required protein phosphatase 1/2A activity and extracellular Ca2+. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na+ or Ca2+ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/ threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways. Keywords: cyclin-dependent kinase 5, depolarization, dopamine-and cAMP-regulated phosphoprotein, N-methyl-D-aspartate, protein phosphatase, protein phosphatase inhibitor-1

Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission

Glutamate counteracts Dopamine/PKA signaling via dephosphorylation of DARPP-32 Ser-97 and alteration of its cytonuclear distribution

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels

PSD volume and its correlation with spine volume.

<p>(A) Scatter plot showing the PSD volume in mice treated with placebo (n = 173 PSDs from 9 dendrites, 3 dendrites per each of 3 mice) or fluoxetine (n = 160 PSDs from 9 dendrites, 3 dendrites per each of 3 mice). (B) Mean values of the PSD volume in the placebo- and fluoxetine-treated mice. *<i>p</i> < 0.05 compared with placebo-treated mice; Mann-Whitney <i>U</i>-test (U = 11920, p = 0.0284). (C) Correlation between the PSD volume and spine volume in the placebo or fluoxetine-treated mice. The fitted lines for the mice treated with placebo (r² = 0.63, <i>p</i> < 0.0001) or fluoxetine (r² = 0.64, <i>p</i> < 0.0001) were obtained through a linear regression analysis.</p

Obligatory roles of dopamine D1 receptors in the dentate gyrus in antidepressant action of a selective serotonin reuptake inhibitor, fluoxetine

Depression is a leading cause of disability. Current pharmacological treatment of depression is insufficient, and development of improved treatments especially for treatment-resistant depression is desired. Understanding the neurobiology of antidepressant actions may lead to development of improved therapeutic approaches. Here, we demonstrate that dopamine D1 receptors in the dentate gyrus act as a pivotal mediator of antidepressant actions in mice. Chronic administration of a selective serotonin reuptake inhibitor (SSRI), fluoxetine, increases D1 receptor expression in mature granule cells in the dentate gyrus. The increased D1 receptor signaling, in turn, contributes to the actions of chronic fluoxetine treatment, such as suppression of acute stress-evoked serotonin release, stimulation of adult neurogenesis and behavioral improvement. Importantly, under severely stressed conditions, chronic administration of a D1 receptor agonist in conjunction with fluoxetine restores the efficacy of fluoxetine actions on D1 receptor expression and behavioral responses. Thus, our results suggest that stimulation of D1 receptors in the dentate gyrus is a potential adjunctive approach to improve therapeutic efficacy of SSRI antidepressants. © 2018, The Author(s).1

Three-dimensional reconstruction of perforant path-GC synapses in the dentate gyrus.

<p>(A, B) Full field SEM images obtained through FIB/SEM show the cross-sections of dendrites (green) in the placebo- (A) and fluoxetine (B)-treated mice. Insets show images of dendritic spines (green) and connecting boutons obtained in other sections. The arrows (red) indicate PSD. (C, D) 3D-reconstructed dendritic segments in the middle molecular layer of the DG in the placebo- (C) and fluoxetine (D)-treated mice. Note the appearance of the large-sized spines and PSDs (red) in the fluoxetine-treated mice. The dendritic spines, which are shown in the insets of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.g001" target="_blank">Fig 1A and 1B</a>, are indicated with numbers. (E, F) Three-dimensional reconstructed presynaptic boutons are visualized at two synapses in the placebo- (E) and fluoxetine (F)-treated mice. The synaptic vesicles (orange) and mitochondria (purple) are shown inside the presynaptic boutons. Note that the large-sized spines are in contact with large-sized presynaptic boutons. Scale bars: 1 μm.</p

Effect of chronic fluoxetine treatment on optical responses in the hippocampal DG.

<p>(A) Effect of chronic fluoxetine treatment on optical responses evoked by the stimulation of the perforant path inputs in hippocampal slices. The left-most panel shows the pseudocolor image of the slice preparation in which the optical recordings were made. A series of optical images of neuronal activity were recorded at 1.2-ms intervals from 0 to 10.8 ms after nerve stimulation. The signal intensity, expressed as fractional changes in optical absorbance relative to the background (%), was coded by the pseudocolor image. (B, C) The maximum propagation area of the optical signal above the background noise was analyzed 7.2–9.6 ms after stimulation, when the activation of the dendrites of the GCs via glutamatergic synaptic transmission was detected (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.s001" target="_blank">S1 Fig</a>). Typical images of the excitation area (B), traces of the optical responses at the boxed area (B) and the quantified excitation area (C) are shown in mice treated with placebo (n = 16 slices from 8 mice) and fluoxetine (n = 19 slices from 9 mice). *<i>p</i> < 0.001 compared with placebo-treated mice; Mann-Whitney <i>U</i>-test (U = 50, p = 0.0008). The preliminary data used for these figures are reported in a review article in Japanese [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147307#pone.0147307.ref071" target="_blank">71</a>].</p

Spine volume and density in the placebo- and fluoxetine-treated mice.

<p>(A) The scatter plot shows the spine volume in mice treated with placebo (n = 207 spines from 9 dendrites, 3 dendrites per each of 3 mice) or fluoxetine (n = 175 spines from 9 dendrites, 3 dendrites per each of 3 mice). (B, C) Mean values of spine volume for all spines (Mann-Whitney <i>U</i>-test: U = 15930, p = 0.0425) (B) and spine density (Mann-Whitney <i>U</i>-test: U = 28, p = 0.2973) (C) in the placebo- or fluoxetine-treated mice. *<i>p</i> < 0.05 compared with the placebo-treated mice.</p