Sulfation patterns of glycosaminoglycans encode molecular recognition and activity

Although glycosaminoglycans contribute to diverse physiological processes, an understanding of their molecular mechanisms has been hampered by the inability to access homogeneous glycosaminoglycan structures. Here, we assembled well-defined chondroitin sulfate oligosaccharides using a convergent, synthetic approach that permits installation of sulfate groups at precise positions along the carbohydrate backbone. Using these defined structures, we demonstrate that specific sulfation motifs function as molecular recognition elements for growth factors and modulate neuronal growth. These results provide both fundamental insights into the role of sulfation and direct evidence for a 'sulfation code' whereby glycosaminoglycans encode functional information in a sequence-specific manner analogous to that of DNA, RNA and proteins

Activation of phospholipase C pathways by a synthetic chondroitin sulfate-E tetrasaccharide promotes neurite outgrowth of dopaminergic neurons

In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS. CS-E polysaccharide, but not CSA, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase ζ and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca^(2+) concentration-dependent activation of Ca^(2+)/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons

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

Upregulation of the dorsal raphe nucleus-prefrontal cortex serotonin system by chronic treatment with escitalopram in hyposerotonergic Wistar-Kyoto rats

<p>Wistar-Kyoto (WKY) rats are sensitive to chronic stressors and exhibit depression-like behavior. Dorsal raphe nucleus (DRN) serotonin (5-HT) neurons projecting to the prefrontal cortex (PFC) comprise the important neurocircuitry underlying the pathophysiology of depression. To evaluate the DRN-PFC 5-HT system in WKY rats, we examined the effects of escitalopram (ESCIT) on the extracellular 5-HT level in comparison with Wistar rats using dual-probe microdialysis. The basal levels of 5-HT in the DRN, but not in the PFC, in WKY rats was reduced as low as 30% of Wistar rats. Responses of 5-HT in the DRN and PFC to ESCIT administered systemically and locally were attenuated in WRY rats. Feedback inhibition of DRN 5-HT release induced by ESCIT into the PFC was also attenuated in WKY rats. Chronic ESCIT induced upregulation of the DRN-PFC 5-HT system in WRY rats, with increases in basal 5-HT in the DRN, responsiveness to ESCIT in the DRN and PFC, and feedback inhibition, whereas downregulation of these effects was induced in Wistar rats. Thus, the WRY rat is an animal model of depression with low activity of the DRN-PFC 5HT system. The finding that chronic ESCIT upregulates the 5-HT system in hyposerotonergic WRY rats may contribute to improved understanding of mechanisms of action of antidepressants, especially in depression with 5-HT deficiency. (C) 2013 Elsevier Ltd. All rights reserved.</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

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

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

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

Characterization of presynaptic boutons connected to spines of different sizes.

<p>(A) Presynaptic boutons were classified by their connections to spines with different sizes. The spines were subdivided into four groups (Groups 1–4) based on the mean and SD values of the spine volume in placebo-treated mice. Typical images of SEM and 3D reconstructed spines (green) and connected boutons (light brown) are shown. Synaptic vesicles (orange) and mitochondria (purple) are shown inside the presynaptic boutons. Scale bars: 1 μm. (B) Volume of presynaptic boutons connected to spines classified as Groups 1–4. n = 6–10 presynaptic boutons in each group. No significant difference between placebo and fluoxetine with two-way ANOVA (drug effect, F_(1,44) = 0.0682, p < 0.794; group effect, F_(3,44) = 11.5, <i>p</i> < 0.0001; drug and group interaction, F_(3,44) = 0.230, <i>p</i> = 0.875). One-way ANOVA: placebo, F_(3,20) = 4.48, <i>p</i> = 0.0147; fluoxetine, F_(3,24) = 7.81, <i>p</i> = 0.0008; Bonferroni’s post-hoc test, *<i>p</i> < 0.05, compared with Group 1 in placebo, ^†<i>p</i> < 0.05 compared with Group 2 in placebo, **<i>p</i> < 0.01 compared with Group 1 in fluoxetine, ^††<i>p</i> < 0.01 compared with Group 2 in fluoxetine, ^§<i>p</i> < 0.05 compared with Group 3 in fluoxetine. (C) Correlation between volumes of mitochondria and presynaptic boutons in mice treated with placebo (r² = 0.79, <i>p</i> < 0.0001) or fluoxetine (r² = 0.84, <i>p</i> < 0.0001) with linear regression analysis. The symbol indicates the number of mitochondrion in each presynaptic bouton: ●, one; ○, two; ▲, three. The mitochondrial volume is the sum of the volume for all of the mitochondria in each bouton. (D) Correlation between the volumes of synaptic vesicles and presynaptic boutons in mice treated with placebo (r² = 0.91, <i>p</i> < 0.0001) or fluoxetine (r² = 0.72, <i>p</i> < 0.0001) with linear regression analysis.</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