Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs.

Eukaryotes respond to secreted metabolites from the microbiome. However, little is known about the effects of exposure to volatiles emitted by microbes or in the environment that we are exposed to over longer durations. Using Drosophila melanogaster, we evaluated a yeast-emitted volatile, diacetyl, found at high levels around fermenting fruits where they spend long periods of time. Exposure to the diacetyl molecules in headspace alters gene expression in the antenna. In vitro experiments demonstrated that diacetyl and structurally related volatiles inhibited conserved histone deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure caused modulation of gene expression in the mouse brain, therefore showing potential as a neuro-therapeutic. Using two separate disease models previously known to be responsive to HDAC inhibitors, we evaluated the physiological effects of volatile exposure. Diacetyl exposure halted proliferation of a neuroblastoma cell line in culture. Exposure to diacetyl vapors slowed progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression, and physiology in animals.

Hypothalamic representation of the imminence of predator threat detected by the vomeronasal organ in mice.

Animals have the innate ability to select optimal defensive behavioral outputs with an appropriate intensity in response to predator threat in specific contexts. Such innate behavioral decisions are thought to be computed in the medial hypothalamic nuclei that contain neural populations directly controlling defensive behavioral outputs. The vomeronasal organ (VNO) is one of the major sensory input channels through which predator cues are detected with ascending inputs to the medial hypothalamic nuclei, especially to the ventromedial hypothalamus (VMH), through the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST). Here, we show that cat saliva contains predator cues that signal imminence of predator threat and regulate the robustness of freezing behavior through the VNO in mice. Cat saliva activates neurons expressing the V2R-A4 subfamily of sensory receptors, suggesting the existence of specific receptor groups responsible for freezing behavior induced by the predator cues. The number of VNO neurons activated in response to saliva correlates with the freshness of saliva and the intensity of freezing behavior, while the downstream neurons in the accessory olfactory bulb (AOB) and defensive behavioral circuit are quantitatively equally activated by fresh and old saliva. Strikingly, however, only the number of VMH neurons activated by fresh saliva positively correlates with the intensity of freezing behavior. Detailed analysis of the spatial distribution of fresh and old saliva-responding neurons revealed a neuronal population within the VMH that is more sensitive to fresh saliva than old saliva. Taken together, this study demonstrates that predator cues in cat saliva change over time and differentially activate the sensory-to-hypothalamus defensive behavioral pathway to modulate behavioral outputs.

Plasticity of gene expression in the nervous system by exposure to environmental odorants that inhibit HDACs.

Eukaryotes are often exposed to microbes and respond to their secreted metabolites, such as the microbiome in animals or commensal bacteria in roots. Little is known about the effects of long-term exposure to volatile chemicals emitted by microbes, or other volatiles that we are exposed to over a long duration. Using the model system Drosophila melanogaster, we evaluate a yeast emitted volatile, diacetyl, found in high levels around fermenting fruits where they spend long periods of time. We find that exposure to just the headspace containing the volatile molecules can alter gene expression in the antenna. Experiments showed that diacetyl and structurally related volatile compounds inhibited human histone-deacetylases (HDACs), increased histone-H3K9 acetylation in human cells, and caused wide changes in gene expression in both Drosophila and mice. Diacetyl crosses the blood-brain barrier and exposure causes modulation of gene expression in the brain, therefore has potential as a therapeutic. Using two separate disease models known to be responsive to HDAC-inhibitors, we evaluated physiological effects of volatile exposure. First, we find that the HDAC inhibitor also halts proliferation of a neuroblastoma cell line in culture as predicted. Next, exposure to vapors slows progression of neurodegeneration in a Drosophila model for Huntington's disease. These changes strongly suggest that unbeknown to us, certain volatiles in the surroundings can have profound effects on histone acetylation, gene expression and physiology in animals.

Age-Related Oxidative Redox and Metabolic Changes Precede Intraneuronal Amyloid-ß Accumulation and Plaque Deposition in a Transgenic Alzheimer's Disease Mouse Model.

BACKGROUND: Many identified mechanisms could be upstream of the prominent amyloid-ß (Aß) plaques in Alzheimer's disease (AD). OBJECTIVE: To profile the progression of pathology in AD. METHODS: We monitored metabolic signaling, redox stress, intraneuronal amyloid-ß (iAß) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan. RESULTS: Intracellular accumulation of aggregated Aß in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAß. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAß and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAß accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine. CONCLUSION: The order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by a pAkt/tAkt metabolic shift in CA1, iAß accumulation in CA1, and extracellular Aß deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes.

Coadaptation of the chemosensory system with voluntary exercise behavior in mice.

Ethologically relevant chemical senses and behavioral habits are likely to coadapt in response to selection. As olfaction is involved in intrinsically motivated behaviors in mice, we hypothesized that selective breeding for a voluntary behavior would enable us to identify novel roles of the chemosensory system. Voluntary wheel running (VWR) is an intrinsically motivated and naturally rewarding behavior, and even wild mice run on a wheel placed in nature. We have established 4 independent, artificially evolved mouse lines by selectively breeding individuals showing high VWR activity (High Runners; HRs), together with 4 non-selected Control lines, over 88 generations. We found that several sensory receptors in specific receptor clusters were differentially expressed between the vomeronasal organ (VNO) of HRs and Controls. Moreover, one of those clusters contains multiple single-nucleotide polymorphism loci for which the allele frequencies were significantly divergent between the HR and Control lines, i.e., loci that were affected by the selective breeding protocol. These results indicate that the VNO has become genetically differentiated between HR and Control lines during the selective breeding process. Although the role of the vomeronasal chemosensory receptors in VWR activity remains to be determined, the current results suggest that these vomeronasal chemosensory receptors are important quantitative trait loci for voluntary exercise in mice. We propose that olfaction may play an important role in motivation for voluntary exercise in mammals.

Signaling Mode of the Broad-Spectrum Conserved CO2 Receptor Is One of the Important Determinants of Odor Valence in Drosophila.

Odor detection involves hundreds of olfactory receptors from diverse families, making modeling of hedonic valence of an odorant difficult, even in Drosophila melanogaster where most receptors have been deorphanised. We demonstrate that a broadly tuned heteromeric receptor that detects CO2 (Gr21a, Gr63a) and other odorants is a key determinant of valence along with a few members of the Odorant receptor family in a T-maze, but not in a trap assay. Gr21a and Gr63a have atypically high amino acid conservation in Dipteran insects, and they use both inhibition and activation to convey positive or negative valence for numerous odorants. Inhibitors elicit a robust Gr63a-dependent attraction, while activators, strong aversion. The attractiveness of inhibitory odorants increases with increasing background CO2 levels, providing a mechanism for behavior modulation in odor blends. In mosquitoes, valence is switched and activation of the orthologous receptor conveys attraction. Reverse chemical ecology enables the identification of inhibitory odorants to reduce attraction of mosquitoes to skin.

Cofilin under control of ß-arrestin-2 in NMDA-dependent dendritic spine plasticity, long-term depression (LTD), and learning.

Dendritic spines are dynamic, actin-rich structures that form the postsynaptic sites of most excitatory synapses in the brain. The F-actin severing protein cofilin has been implicated in the remodeling of dendritic spines and synapses under normal and pathological conditions, by yet unknown mechanisms. Here we report that ß-arrestin-2 plays an important role in NMDA-induced remodeling of dendritic spines and synapses via translocation of active cofilin to dendritic spines. NMDAR activation triggers cofilin activation through calcineurin and phosphatidylinositol 3-kinase (PI3K)-mediated dephosphorylation and promotes cofilin translocation to dendritic spines that is mediated by ß-arrestin-2. Hippocampal neurons lacking ß-arrestin-2 develop mature spines that fail to remodel in response to NMDA. ß-Arrestin-2-deficient mice exhibit normal hippocampal long-term potentiation, but significantly impaired NMDA-dependent long-term depression and spatial learning deficits. Moreover, ß-arrestin-2-deficient hippocampal neurons are resistant to Aß-induced dendritic spine loss. Our studies demonstrate unique functions of ß-arrestin-2 in NMDAR-mediated dendritic spine and synapse plasticity through spatial control over cofilin activation.

Focal adhesion kinase acts downstream of EphB receptors to maintain mature dendritic spines by regulating cofilin activity.

Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain and are highly enriched in polymerized F-actin, which drives the formation and maintenance of mature dendritic spines and synapses. We propose that suppressing the activity of the actin-severing protein cofilin plays an important role in the stabilization of mature dendritic spines, and is accomplished through an EphB receptor-focal adhesion kinase (FAK) pathway. Our studies revealed that Cre-mediated knock-out of loxP-flanked fak prompted the reversion of mature dendritic spines to an immature filopodial-like phenotype in primary hippocampal cultures. The effects of FAK depletion on dendritic spine number, length, and morphology were rescued by the overexpression of the constitutively active FAK(Y397E), but not FAK(Y397F), indicating the significance of FAK activation by phosphorylation on tyrosine 397. Our studies demonstrate that FAK acts downstream of EphB receptors in hippocampal neurons and EphB2-FAK signaling controls the stability of mature dendritic spines by promoting cofilin phosphorylation, thereby inhibiting cofilin activity. While constitutively active nonphosphorylatable cofilin(S3A) induced an immature spine profile, phosphomimetic cofilin(S3D) restored mature spine morphology in neurons with disrupted EphB activity or lacking FAK. Further, we found that EphB-mediated regulation of cofilin activity at least partially depends on the activation of Rho-associated kinase (ROCK) and LIMK-1. These findings indicate that EphB2-mediated dendritic spine stabilization relies, in part, on the ability of FAK to activate the RhoA-ROCK-LIMK-1 pathway, which functions to suppress cofilin activity and inhibit cofilin-mediated dendritic spine remodeling.

Accelerators, Brakes, and Gears of Actin Dynamics in Dendritic Spines.

Dendritic spines are actin-rich structures that accommodate the postsynaptic sites of most excitatory synapses in the brain. Although dendritic spines form and mature as synaptic connections develop, they remain plastic even in the adult brain, where they can rapidly grow, change, or collapse in response to normal physiological changes in synaptic activity that underlie learning and memory. Pathological stimuli can adversely affect dendritic spine shape and number, and this is seen in neurodegenerative disorders and some forms of mental retardation and autism as well. Many of the molecular signals that control these changes in dendritic spines act through the regulation of filamentous actin (F-actin), some through direct interaction with actin, and others via downstream effectors. For example, cortactin, cofilin, and gelsolin are actin-binding proteins that directly regulate actin dynamics in dendritic spines. Activities of these proteins are precisely regulated by intracellular signaling events that control their phosphorylation state and localization. In this review, we discuss how actin-regulating proteins maintain the balance between F-actin assembly and disassembly that is needed to stabilize mature dendritic spines, and how changes in their activities may lead to rapid remodeling of dendritic spines.

Regulation of tryptophan hydroxylase-2 gene expression by a bipartite RE-1 silencer of transcription/neuron restrictive silencing factor (REST/NRSF) binding motif.

Tryptophan hydroxylase-2 (TPH2) is the rate-limiting enzyme in raphe serotonin biosynthesis, and polymorphisms of TPH2 are implicated in vulnerability to psychiatric disorders. Dynamic transcription regulation of TPH2 may underlie differences in vulnerability. We identified a transcription element in the TPH2 promoter that resembles the binding motif for RE-1 silencer of transcription (REST; also known as NRSF) transcription factor. REST limits tissue expression of non-neuronal genes through a canonical 21-bp motif called the NRSE (neuron-restrictive silencing element). The NRSE in TPH2 is a novel bipartite variant interrupted by a 6-base insertion. We confirmed that this bipartite NRSE permits transcriptional repression by REST identical to canonical NRSE in rat C6-glioma cells. Synthetic permutations of the motif revealed considerable flexibility in the juxtaposition of the two halves of bipartite NRSE. Computational analysis revealed many bipartite NRSE variants conserved between mouse and human genomes. A subgroup of these was found to bind REST by chromatin immunoprecipitation. Messenger RNAs for TPH2 and potassium channel H6, another gene with a bipartite NRSE, were up-regulated by dominant-negative REST in C6-glioma cells. These findings, which indicate that TPH2 expression is part of the developmental program regulated by REST and suggest that many previously unrecognized genes may be regulated by REST through the novel motif, have implications for the mechanism of REST action.

Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland.

BACKGROUND: Regulation of raphe serotonergic cells is fundamental to the prevailing hypothesis of major depression pathophysiology. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis, but brainstem TPH mRNA expression has been difficult to measure and study. Recently, a novel paralog of TPH, TPH2 (or neuronal TPH), was described, but its anatomic expression is unknown. METHODS: In situ hybridization histochemical survey was conducted across Sprague-Dawley rat brain for TPH1 and TPH2 mRNA. Semiquantitative techniques were used to estimate relative mRNA levels in individual cells. RESULTS: Almost exclusively, TPH2 mRNA is expressed in raphe, in a pattern overlapping the histologically defined raphe nuclei. In sharp contrast, TPH1 (the previously known TPH) is expressed predominantly in pineal gland. There is no appreciable overlap in the expression of these paralogs. The level of TPH2 mRNA expression in individual raphe cells is approximately 2.5-fold greater than the level of TPH1 expression in pinealocytes. CONCLUSIONS: TPH2 mRNA has an anatomic expression pattern consistent with brainstem raphe nuclei and is likely to be the gene giving rise to the majority of TPH activity in these cells. The robust expression of TPH2 in brainstem should facilitate studies on the transcriptional regulation of raphe serotonin biosynthesis.