Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system

Choreographing the axo-dendritic dance

The assembly of neuronal synapses in the brain relies on a sophisticated bidirectional signal exchange between synaptic partners. In a recent issue of Neuron, Ito-Ishida and colleagues (2012) uncover a morphogenetic program underlying the formation of presynaptic terminals

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

<div><p>Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (<i>N</i>-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.</p></div

Bidirectional regulation of enhanced GABA release via chronic changes in neuronal activity.

<p>(A) Cultured cortical neurons treated with 50 μM bicuculline or 1 μM TTX on DIV14 or on DIV12, respectively, were depolarized with 4-AP on DIV15 to test enhanced neurotransmitter release. (B) Enhanced GABA release following chronic changes in neuronal activity. F(2, 6) = 101.0 (<i>p</i> < .0001), one-way ANOVA. (C) Enhanced glutamate release following chronic changes in neuronal activity. F(2, 6) = 0.46 (no significant difference, <i>p</i> = 0.7), one-way ANOVA. The right trace in (B) and (C) shows the representative chromatographic peaks of neurotransmitters on HPLC in the representative experiment. Data were analyzed from 3 independent cultures.</p

Released BDNF differentially accelerates expression of GADs under conditions of increased activity.

<p>(A) Cultured cortical neurons were treated with 50 μM bicuculline on DIV14 for the final day in the culture, and the conditioned medium was harvested on DIV15. (B) Level of released BDNF from cultured cortical neurons after treatment with bicuculine as shown in (A). <i>(n</i> = 6) Arrow shows mature BDNF. Note that the asterisk is an unknown, unspecific band (not pro-BDNF) because a previous study used the same antibody that was detected in BDNF knockout mice. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134296#pone.0134296.ref022" target="_blank">22</a>] (C) Recombinant human BDNF (100 ng/mL) is applied to cultured cortical neurons for 24 hours with or without inhibitors. (D) Relative GAD protein levels after treatment with BDNF. The increased expression of GAD65 and GAD67 is inhibited by K252a but not by KN93. Note that BDNF preferentially enhances GAD65 expression over that of GAD67. GAD67: F(3, 38) = 19.35 (<i>p</i> < .0001); GAD65: F(3, 25) = 21.78 (<i>p</i> < .0012), one-way ANOVA. <i>(n</i> = 6–10 in each group).</p

Proposed model of activity-dependent GAD expression through BDNF-dependent and independent pathways triggered by NMDA-R activation.

<p>The basal level of GAD expression is largely determined by spontaneous NMDA-R activity on GABAergic neurons. When neuronal activity is persistently increased, expression of both GAD isoforms is up-regulated by increased Ca²⁺ influx into the GABAergic neurons. In parallel, activity-dependent expression of BDNF is also induced by NMDA-R activation through CaMK and MAPK in neighboring excitatory neurons. Activity-regulated BDNF subsequently accelerates GAD expression through TrkB-MAPK signaling in GABAergic neurons. With increased activity, GAD65 is preferentially up regulated by a BDNF-dependent and non-cell autonomous mechanism (right). Conversely, when neuronal activity is silenced, the attenuated Ca²⁺ level in GABAergic neurons leads to a reduction in basal GAD expression. Activity deprivation has a stronger effect on the expression of GAD67 than of GAD65. Overall, we speculate that GABA synthesis may be bidirectionally regulated in a homeostatic fashion by this activity-dependent expression of two GAD isoforms through the interplay of BDNF-dependent and independent pathways triggered by NMDA-R activation.</p

Activity deprivation reduces GAD expression with NMDA-R-dependent and BDNF-independent regulation.

<p>(A) Cultured cortical neurons are treated with bicuculline (50 μM), TTX (1 μM), K252a (100 nM), U0126 (10 μM), or AP5 (200 μM) for the last 3 days in a culture and are harvested on DIV15. (B) Relative GAD65 mRNA expression levels in neurons treated as shown in (A). The basal expression level of GAD65 is selectively reduced by inactivity caused by TTX or AP5 but not by the blockade of BDNF-TrkB signaling with K252a or U0126. F(5, 56) = 40.42 (<i>p</i> < .0001) one-way ANOVA. (<i>n</i> = 4–6 in each group). (C) Cultured cortical neurons are treated with TTX (1 μM), d,l-AP5 (200 μM), or co-treated with TTX and d,l-AP5 (200 μM) on DIV12 and are harvested on DIV15. (D) Relative GAD mRNA expression levels in cultured cortical neurons treated as shown in (C). The inhibitory effect of long-term TTX on GAD expression is completely occluded by AP5. GAD67 shows significantly decreased expression in TTX-treated neurons compared with GAD65. GAD67: F(2, 9) = 206.5; GAD65: F (2, 9) = 79.05 (<i>p</i> < .0001), one-way ANOVA. (<i>n</i> = 4 in each group). (E) Representative images of endogenous GAD67 and vGluT1 protein immunocytochemistry in TTX- and TTX plus AP5-treated cultures. Cultured cortical neurons are triple-stained with anti-GAD67, anti-vGluT1, and MAP2 antibodies. (F) Intensities of GAD67 and vGluT1 immunoreactivity measured in MAP2-positive dendritic areas. Co-application of TTX and AP5 does not have an additive effect on reducing GAD67 immunoreactivity. Twelve confocal images in each condition were analyzed from 3 independent experiments. GAD67: F(2, 23) = 14.40 (<i>p</i> < .0001); vGluT1: F(2, 33) = 1.152 (no significant difference, <i>p</i> = 0.3), one-way ANOVA. Scale bar = 50 μm.</p

Different bidirectional forms of activity-dependent gene regulation governing GAD expression.

<p>(A) Cultured cortical neurons were treated with 50 μM bicuculline on DIV14 or with 1 μM TTX on DIV12 and were harvested on DIV15. (B) Shows reciprocal effects of bicuculline and TTX on relative expression levels of mRNAs encoding GABAergic and glutamatergic presynaptic proteins in cultured cortical neurons treated as shown in (A). GAD67: F(2, 14) = 106.5 (<i>p</i> < .0001); GAD65: F (2, 14) = 62.36 (<i>p</i> < .0001); vGluT1: F(2, 14) = 11.52 (<i>p</i> = 0.001); VAMP2: F(2, 14) = 1.26 (no significant difference, <i>p</i> = 0.31), one-way ANOVA. (<i>n</i> = 5–6 in each group). GAD: glutamic acid decarboxylase; DIV: days in vitro; TTX: tetrodotoxin.</p

Distinct Defects in Synaptic Differentiation of Neocortical Neurons in Response to Prenatal Valproate Exposure

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders characterized by impairments in social interactions and stereotyped behaviors. Valproic acid (VPA) is frequently used to treat epilepsy and bipolar disorders. When taken during pregnancy, VPA increases the risk of the unborn child to develop an ASD. In rodents, in utero VPA exposure can precipitate behavioral phenotypes related to ASD in the offspring. Therefore, such rodent models may allow for identification of synaptic pathophysiology underlying ASD risk. Here, we systematically probed alterations in synaptic proteins that might contribute to autism-related behavior in the offspring of in utero VPA-exposed mice. Moreover, we tested whether direct VPA exposure of cultured neocortical neurons may recapitulate the molecular alterations seen in vivo. VPA-exposed neurons in culture exhibit a significant increase in the number of glutamatergic synapses accompanied by a significant decrease in the number of GABAergic synapses. This shift in excitatory/inhibitory balance results in substantially increased spontaneous activity in neuronal networks arising from VPA-exposed neurons. Pharmacological experiments demonstrate that the alterations in GABAergic and glutamatergic synaptic proteins and structures are largely caused by inhibition of histone deacetylases. Therefore, our study highlights an epigenetic mechanism underlying the synaptic pathophysiology in this ASD model